TW201615832A - Treatment of cancer using humanized anti-BCMA chimeric antigen receptor - Google Patents

Abstract

The invention provides compositions and methods for treating diseases associated with expression of BCMA. The invention also relates to chimeric antigen receptor (CAR) specific to BCMA vectors encoding the same, and recombinant T cells comprising the BCMA CAR. The invention also includes methods of administering a genetically modified T cell expressing a CAR that comprises a BCMA binding domain.

Description

Treatment of cancer with a humanized anti-BCMA chimeric antigen receptor

The priority of PCT Application No. PCT/CN2014/090501, filed on Nov. 6, 2014, and PCT Application No. PCT/CN2014/082586, filed on Jul. 21, 2014. The entire contents of these applications are hereby incorporated by reference.

Sequence table

This application contains a Sequence Listing which has been filed electronically in ASCII format and the entire contents of which are incorporated herein by reference. The ASCII copy generated on July 15, 2015 is named N2067-7045WO3_SL.txt and has a size of 771,026 bytes.

The present invention relates generally to the use of immune effector cells (e.g., T cells, NK cells) engineered to exhibit chimeric antigen receptors (CARs) for use in treating the expression of B cell mature antigen protein (BCMA) disease.

B cell maturation antigen (BCMA) is a member of the tumor necrosis family receptor (TNFR) that is expressed by cells of the B cell lineage. BCMA was the highest on terminally differentiated B cells. BCMA is involved in mediating the survival of plasma cells to maintain long-term humoral immunity. The performance of BCMA has recently been associated with many cancers, autoimmune disorders and infectious diseases. Cancers with increased BCMA include some blood cancers such as multiple myeloma, Hodgkin's lymphoma and non-Hodgkin's lymphoma, various leukemias and glia Cell tumor.

In a first aspect, the invention relates to an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises a human anti-BCMA binding domain or a humanized anti-BCMA binding domain, An antibody or antibody fragment that is a transmembrane domain and an intracellular signaling domain (eg, an intracellular signaling domain comprising a costimulatory domain and/or a major signaling domain). In one embodiment, the CAR comprises a human anti-BCMA binding domain as described herein or a humanized anti-BCMA binding domain as described herein, a transmembrane domain as described herein, and an intracellular signal as described herein. A transduction domain (eg, an antibody or antibody fragment comprising a co-stimulatory domain and/or an intracellular signaling domain of a major signaling domain).

In one embodiment, the encoded BCMA binding domain (eg, a human or humanized anti-BCMA binding domain) comprises one or more of the human or humanized anti-BCMA binding domains described herein (eg, all three) Light chain complementarity determining region 1 (LC CDR1), light chain complementarity determining region 2 (LC CDR2) and light chain complementarity determining region 3 (LC CDR3), and/or one of the anti-BCMA binding domains described herein Or multiple (eg, all three) heavy chain complementarity determining region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2), and heavy chain complementarity determining region 3 (HC CDR3), for example, comprising one or more, eg, All three LC CDRs and one or more, such as human or humanized anti-BCMA binding domains of all three HC CDRs. In one embodiment, the encoded human anti-BCMA binding domain comprises a light chain variable region as described herein (eg, in Table 1) and/or a heavy chain variable region as described herein (eg, in Table 1) . In one embodiment, the encoded humanized anti-BCMA binding domain comprises the light chain variable region provided in SEQ ID NO: 271 or 273 and/or the heavy chain provided in SEQ ID NO: 271 or 273 Variable zone. In one embodiment, the encoded human anti-BCMA binding domain is a scFv comprising the light and heavy chains of the amino acid sequence of Table 1. In one embodiment, the encoded humanized anti-BCMA binding domain is a scFv comprising the light and heavy chains of the amino acid sequence of SEQ ID NO: 271 or 273. In one embodiment, the human or humanized anti-BCMA binding domain (eg, scFv) comprises: comprising an amino acid sequence having a light chain variable region as provided in Table 1 or SEQ ID NO: 271 or 273 An amino acid sequence of at least one, two or three modifications (eg, substitutions, eg, conservative substitutions) but no more than 30, 20 or 10 modifications (eg, substitutions, eg, conservative substitutions), or with Table 1 or SEQ ID NO: The amino acid sequence of 271 or 273 has a light chain variable region of 95%-99% identity; and/or comprises a heavy chain as provided in Table 1 or SEQ ID NO: 271 or 273 Amino acid sequence of at least one, two or three modifications (eg, substitutions, eg, conservative substitutions) of the amino acid sequence of the variable region but no more than 30, 20 or 10 modifications (eg, substitutions, eg, conservative substitutions) Or a heavy chain variable region of the sequence having 95%-99% identity to the amino acid sequence of Table 1. In one embodiment, the encoded human anti-BCMA binding domain comprises selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO :52, SEQ ID NO: 53, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135 SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ a sequence of a population consisting of ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, and SEQ ID NO: 149, or 95%-99% identical thereto The sequence. In one embodiment, the encoded humanized anti-BCMA binding domain comprises a sequence selected from the group consisting of SEQ ID NO:271 or SEQ ID NO:273, or a sequence having 95%-99% identity thereto. In one embodiment, the encoded human or humanized anti-BCMA binding domain comprises a (Gly ₄ -Ser) n linker, wherein n is 1, 2, 3, 4, 5 or 6, preferably 3 or 4 (SEQ ID NO: 26). The light chain variable region and the heavy chain variable region of the scFv can, for example, be in any of the following orientations: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain Variable zone.

In other embodiments, the encoded BCMA binding domain comprises the HC CDR1, HC CDR2, and HC CDR3 of any of the BCMA heavy chain binding domain amino acid sequences set forth in Tables 1 or 16. In an embodiment, the BCMA binding domain further comprises LC CDR1, LC CDR2, and LC CDR3. In an embodiment, the BCMA binding domain comprises the LC CDR1, LC CDR2 and LC CDR3 of any of the BCMA light chain binding domain amino acid sequences listed in Table 1 or 16.

In some embodiments, the encoded BCMA binding domain comprises one of LC CDR1, LC CDR2 and LC CDR3 of any of the BCMA light chain binding domain amino acid sequences listed in Table 1 or 16 or both All, and one, both or all of the HC CDR1, HC CDR2 and HC CDR3 of any of the BCMA heavy chain binding domain amino acid sequences listed in Table 1 or 16.

In one embodiment, the encoded anti-BCMA binding domain comprises a light chain variable region as described herein (eg, in Table 16) and/or a heavy chain variable region (eg, in Table 16) as described herein. In one embodiment, the encoded humanized anti-BCMA binding domain comprises the light chain variable region provided in SEQ ID NO:259, SEQ ID NO:260, SEQ ID NO:261, SEQ ID NO:262 And/or the heavy chain variable region provided in SEQ ID NO: 255, SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258. In one embodiment, the encoded anti-BCMA binding domain is a scFv comprising the light and heavy chains of the amino acid sequence of Table 16. In one embodiment, the human or humanized anti-BCMA binding domain (eg, scFv) comprises: comprising the pair of SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 261, SEQ ID NO: Providing at least one, two or three modifications (eg, substitutions, eg, conservative substitutions) of the amino acid sequence of the light chain variable region but no more than 30, 20 or 10 modifications (eg, substitutions, such as conservative substitutions) An amino acid sequence, or a light chain variable region thereof having a sequence with 95%-99% identity; and/or comprising a pair of SEQ ID NO: 255, SEQ ID NO: 256, SEQ ID NO: 257, SEQ At least one, two or three modifications (eg, substitutions, eg, conservative substitutions) of the amino acid sequence of the heavy chain variable region provided in ID NO: 258 but no more than 30, 20 or 10 modifications (eg, Substituting, for example, a conservatively substituted amino acid sequence, or a heavy chain variable region thereof having a sequence that is 95% to 99% identical. In one embodiment, the encoded anti-BCMA binding domain comprises a (Gly ₄ -Ser) n linker, wherein n is 1, 2, 3, 4, 5 or 6, preferably 3 or 4 (SEQ ID NO: 26). The light chain variable region and the heavy chain variable region of the scFv can, for example, be in any of the following orientations: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain Variable zone.

In one embodiment, the encoded human anti-BCMA binding domain comprises a sequence selected from the group consisting of SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 265, and SEQ ID NO: 266, or A sequence with 95%-99% identity.

In one embodiment, the encoded CAR comprises a transmembrane domain comprising, for example, a transmembrane domain of a protein selected from the group consisting of: a cell, a beta or a purine chain of a T cell receptor. , CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154. In one embodiment, the encoded transmembrane domain comprises the sequence of SEQ ID NO: 6. In one embodiment, the encoded transmembrane domain comprises an amine comprising at least one, two or three modifications to the amino acid sequence of SEQ ID NO: 6, but no more than 20, 10 or 5 modifications The acid sequence or sequence having 95% to 99% identity to the amino acid sequence of SEQ ID NO: 6. In one embodiment, the nucleic acid sequence encoding a transmembrane domain comprises the sequence of SEQ ID NO: 17, or a sequence with 95%-99% identity thereto.

In one embodiment, the encoded anti-BCMA binding domain is joined to the transmembrane domain by a hinge region (eg, a hinge region as described herein). In one embodiment, the encoded hinge region comprises SEQ ID NO: 2 or a sequence with 95%-99% identity thereto. In one embodiment, the nucleic acid sequence encoding the hinge region comprises the sequence of SEQ ID NO: 13 or It has a sequence of 95%-99% identity.

In one embodiment, the isolated nucleic acid molecule further comprises a sequence encoding a costimulatory domain, such as the costimulatory domain described herein. In an embodiment, the intracellular signaling domain comprises a costimulatory domain. In an embodiment, the intracellular signaling domain comprises a major signaling domain. In an embodiment, the intracellular signaling domain comprises one or more of a costimulatory domain and a primary signaling domain (eg, one or more, two or more, or three or more).

In one embodiment, the encoded costimulatory domain is a functional signaling domain obtained, for example, from a protein selected from the group consisting of: MHC class I molecules, TNF receptor proteins, Immunoglobulin-like protein, cytokine receptor, integrin, signaling lymphocyte activating molecule (SLAM protein), activated NK cell receptor, BTLA, Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR ), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f , ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (B Y55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP- 76. PAG/Cbp, CD19a and a ligand that specifically binds to CD83. In an embodiment, The co-stimulatory domain encoded includes 4-1BB, CD27, CD28 or ICOS.

In one embodiment, the encoded co-stimulatory domain of 4-1BB comprises the amino acid sequence of SEQ ID NO: 7. In one embodiment, the encoded costimulatory domain comprises an amine having at least one, two or three modifications to the amino acid sequence of SEQ ID NO: 7, but no more than 20, 10 or 5 modifications The acid sequence or the sequence having 95% to 99% identity to the amino acid sequence of SEQ ID NO: 7. In one embodiment, the nucleic acid sequence encoding the costimulatory domain comprises the nucleotide sequence of SEQ ID NO: 18 or a sequence with 95% to 99% identity thereto. In another embodiment, the encoded co-stimulatory domain of CD28 comprises the amino acid sequence of SEQ ID NO: 1104. In one embodiment, the encoded co-stimulatory domain comprises an amine having at least one, two or three modifications to the amino acid sequence of SEQ ID NO: 1104 but no more than 20, 10 or 5 modifications The acid sequence or sequence having 95% to 99% identity to the amino acid sequence of SEQ ID NO: 1104. In one embodiment, the nucleic acid sequence encoding the costimulatory domain of CD28 comprises the nucleotide sequence of SEQ ID NO: 1105 or a sequence with 95%-99% identity thereto. In another embodiment, the encoded co-stimulatory domain of CD27 comprises the amino acid sequence of SEQ ID NO: 8. In one embodiment, the encoded costimulatory domain comprises an amine having at least one, two or three modifications to the amino acid sequence of SEQ ID NO: 8, but no more than 20, 10 or 5 modifications The acid sequence, or a sequence having 95% to 99% identity to the amino acid sequence of SEQ ID NO: 8. In one embodiment, the nucleic acid sequence encoding the costimulatory domain of CD27 comprises the nucleotide sequence of SEQ ID NO: 19 or a sequence with 95%-99% identity thereto. In another embodiment, the encoded co-stimulatory domain of ICOS comprises the amino acid sequence of SEQ ID NO: 1106. In one embodiment, the encoded costimulatory domain comprises an amine having at least one, two or three modifications to the amino acid sequence of SEQ ID NO: 1106 but no more than 20, 10 or 5 modifications The acid sequence or sequence having 95% to 99% identity to the amino acid sequence of SEQ ID NO: 1106. In one embodiment, the nucleic acid sequence encoding the co-stimulatory domain of ICOS comprises The nucleotide sequence of SEQ ID NO: 1107 or a sequence having 95% to 99% identity thereto.

In an embodiment, the encoded major signaling domain comprises a functional signaling domain of CD3ξ. In an embodiment, the functional signaling domain of CD3ξ comprises or is 95%-99% identical to the amino acid sequence of SEQ ID NO: 9 (mutant CD3ζ) or SEQ ID NO: 10 (wild type human CD3ζ) The sequence.

In one embodiment, the encoded intracellular signaling domain comprises a functional signaling domain of 4-1BB and/or a functional signaling domain of CD3ξ. In one embodiment, the encoded intracellular signaling domain of 4-1BB comprises the amino acid sequence of SEQ ID NO: 7 and/or the CD3 glutamic acid sequence of SEQ ID NO: 9 or SEQ ID NO: . In one embodiment, the intracellular signaling domain comprises at least one, two having an amino acid sequence of SEQ ID NO: 7 and/or an amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10. Or three modified but no more than 20, 10 or 5 modified amino acid sequences or with the amino acid sequence of SEQ ID NO: 7 and/or the amino group of SEQ ID NO: 9 or SEQ ID NO: The acid sequence has a sequence of 95%-99% identity. In one embodiment, the encoded intracellular signaling domain comprises the sequence of SEQ ID NO: 7 and the sequence of SEQ ID NO: 9 or SEQ ID NO: 10, wherein the sequences comprising the intracellular signaling domain Expressed in the same framework and in the form of a single polypeptide chain. In one embodiment, the nucleic acid sequence encoding the intracellular signaling domain of 4-1BB comprises the nucleotide sequence of SEQ ID NO: 18 or a sequence thereof that is 95%-99% identical, and/or SEQ ID NO :20 or the CD3 purine nucleotide sequence of SEQ ID NO: 21 or a sequence having 95% to 99% identity thereto.

In one embodiment, the encoded intracellular signaling domain comprises a functional signaling domain of CD27 and/or a functional signaling domain of CD3ξ. In one embodiment, the intracellular signaling domain of the encoded CD27 comprises the amino acid sequence of SEQ ID NO: 8 and/or the CD3 glutamic acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10. In one embodiment, the intracellular signaling domain comprises at least one of the amino acid sequence having SEQ ID NO: 8 and/or the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10. , two or three modifications but no more than 20, 10 or 5 modified amino acid sequences, or with the amino acid sequence of SEQ ID NO: 8 and/or SEQ ID NO: 9 or SEQ ID NO The amino acid sequence of 10 has a sequence of 95%-99% identity. In one embodiment, the encoded intracellular signaling domain comprises the sequence of SEQ ID NO: 8 and the sequence of SEQ ID NO: 9 or SEQ ID NO: 10, wherein the sequences that comprise the intracellular signaling domain Expressed in the same framework and in the form of a single polypeptide chain. In one embodiment, the nucleic acid sequence encoding the intracellular signaling domain of CD27 comprises the nucleotide sequence of SEQ ID NO: 19 or a sequence thereof that is 95%-99% identical, and/or SEQ ID NO: 20. Or the CD3 purine nucleotide sequence of SEQ ID NO: 21 or a sequence having 95% to 99% identity thereto.

In one embodiment, the encoded intracellular signaling domain comprises a functional signaling domain of CD28 and/or a functional signaling domain of CD3ξ. In one embodiment, the encoded intracellular signaling domain of CD28 comprises the amino acid sequence of SEQ ID NO: 1104 and/or the CD3 glutamic acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10. In one embodiment, the intracellular signaling domain comprises at least one, two having the amino acid sequence of SEQ ID NO: 1104 and/or the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10. Or three modified but no more than 20, 10 or 5 modified amino acid sequences or with the amino acid sequence of SEQ ID NO: 1104 and/or the amino group of SEQ ID NO: 9 or SEQ ID NO: 10. The acid sequence has a sequence of 95%-99% identity. In one embodiment, the encoded intracellular signaling domain comprises the sequence of SEQ ID NO: 1104 and the sequence of SEQ ID NO: 9 or SEQ ID NO: 10, wherein the sequences that comprise the intracellular signaling domain Expressed in the same framework and in the form of a single polypeptide chain. In one embodiment, the nucleic acid sequence encoding the intracellular signaling domain of CD28 comprises the nucleotide sequence of SEQ ID NO: 1105 or a sequence having 95%-99% identity thereto, and/or SEQ ID NO:20 Or the CD3 purine nucleotide sequence of SEQ ID NO: 21 or a sequence having 95% to 99% identity thereto.

In one embodiment, the encoded intracellular signaling domain comprises the work of ICOS Functional signaling domain and/or functional signaling domain of CD3ξ. In one embodiment, the encoded intracellular signaling domain of ICOS comprises the amino acid sequence of SEQ ID NO: 1106 and/or the CD3 glutamic acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10. In one embodiment, the intracellular signaling domain comprises at least one, two having the amino acid sequence of SEQ ID NO: 1106 and/or the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10. Or three modified but no more than 20, 10 or 5 modified amino acid sequences or with the amino acid sequence of SEQ ID NO: 1106 and/or the amino group of SEQ ID NO: 9 or SEQ ID NO: The acid sequence has a sequence of 95%-99% identity. In one embodiment, the encoded intracellular signaling domain comprises the sequence of SEQ ID NO: 1106 and the sequence of SEQ ID NO: 9 or SEQ ID NO: 10, wherein the sequences that comprise the intracellular signaling domain Expressed in the same framework and in the form of a single polypeptide chain. In one embodiment, the nucleic acid sequence encoding the intracellular signaling domain of ICOS comprises the nucleotide sequence of SEQ ID NO: 1107 or a sequence having 95%-99% identity thereto, and/or SEQ ID NO:20 Or the CD3 purine nucleotide sequence of SEQ ID NO: 21 or a sequence having 95% to 99% identity thereto.

In another aspect, the invention relates to an isolated nucleic acid molecule encoding a CAR construct comprising: a leader sequence, such as a leader sequence described herein, eg, an amino acid of SEQ ID NO: Sequence; an anti-BCMA binding domain as described herein, such as a human anti-BCMA binding domain comprising LC CDR1, LC CDR2, LC CDR3, HC CDR1, HC CDR2 and HC CDR3 as described herein (eg, Table 1 or 16) a human anti-BCMA binding domain as described herein or a sequence having 95%-99% identity thereto; a hinge region as described herein, such as the amino acid sequence of SEQ ID NO: 2; a transmembrane domain of the sequence of SEQ ID NO: 6; and an intracellular signaling domain, such as the intracellular signaling domain described herein. In one embodiment, the encoded intracellular signaling domain comprises a costimulatory domain, such as a costimulatory domain as described herein (eg, 4-1BB co-stimulation with the amino acid sequence of SEQ ID NO: 7) a domain or a CD27 costimulatory domain having the amino acid sequence of SEQ ID NO: 8), and/or a major signaling domain, such as a major signaling domain as described herein (eg, having SEQ ID NO: 9 or CD3ξ stimulation domain of the sequence of SEQ ID NO: 10). In one embodiment, an isolated nucleic acid molecule encoding a CAR construct comprises a sequence encoding a leader sequence or a 95%-99% identity thereof from the nucleic acid sequence of SEQ ID NO: 1.

In another aspect, the invention relates to an isolated nucleic acid molecule encoding a CAR construct comprising: a leader sequence, such as a leader sequence described herein, eg, an amino acid of SEQ ID NO: Sequence; an anti-BCMA binding domain as described herein (eg, a humanized anti-BCMA binding domain comprising a LC CDR1, LC CDR2, LC CDR3, HC CDR1, HC CDR2 and/or HC CDR3 as described herein, eg, The humanized anti-BCMA binding domain (eg, a humanized anti-BCMA binding domain comprising a sequence selected from the group consisting of SEQ ID NO: 271 or SEQ ID NO: 273) or 95%-99% thereof Sequence of identity; a hinge region as described herein, such as the amino acid sequence of SEQ ID NO: 2; described herein, for example, a transmembrane domain having the sequence of SEQ ID NO: 6; and an intracellular signaling structure A domain, such as the intracellular signaling domain described herein. In one embodiment, the encoded intracellular signaling domain comprises a costimulatory domain, such as a co-stimulatory domain as described herein (eg, a 4-1BB costimulatory domain having the sequence of SEQ ID NO: 7), And/or a major signaling domain, such as a major signaling domain as described herein (eg, a CD3 ξ stimulating domain having the sequence of SEQ ID NO: 9 or SEQ ID NO: 10). In one embodiment, an isolated nucleic acid molecule encoding a CAR construct comprises a sequence encoding a leader sequence or a 95%-99% identity thereof from the nucleic acid sequence of SEQ ID NO: 1.

In one embodiment, the isolated nucleic acid molecule encoding a CAR construct comprises SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO :59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 150, SEQ ID NO : 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159 SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ The human anti-BCMA binding domain sequence encoded by the nucleic acid sequence of ID NO: 168, SEQ ID NO: 169 or SEQ ID NO: 170, or a sequence with 95% to 99% identity thereto. In one embodiment, the isolated nucleic acid molecule encoding a CAR construct comprises a humanized anti-BCMA binding domain sequence encoded by the nucleic acid sequence of SEQ ID NO: 272, SEQ ID NO: 274, or 95%-99 therefrom The sequence of % consistency.

In one embodiment, the isolated nucleic acid molecule comprises (eg, consists of) encoding SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232 or SEQ ID NO: 233 of the CAR amino acid sequence or with SEQ ID NO: 99, SEQ ID NO :100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108 SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225, Amino group of SEQ ID NO:226, SEQ ID NO:227, SEQ ID NO:228, SEQ ID NO:229, SEQ ID NO:230, SEQ ID NO:231, SEQ ID NO:232 or SEQ ID NO:233 The acid sequence has 85%, 90%, 95%, 96%, 97%, 98% or 99% identity or has one, two or three modifications (eg substitutions, eg conservative substitutions) but no more than 30, 20 Nucleic acid of the amino acid sequence of one or ten modifications (eg, substitutions, such as conservative substitutions).

In one embodiment, the isolated nucleic acid molecule comprises (eg, consists of) SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO :119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127 SEQ ID NO: 128, SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO a nucleic acid sequence of: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252, SEQ ID NO: 253 or SEQ ID NO: 254, or SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ I D NO: 128, SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252, SEQ ID NO: 253 or SEQ ID NO: 254 The amino acid sequence of the nucleic acid sequence has 85%, 90%, 95%, 96%, 97%, 98% or 99% identity or has one, two or three modifications (eg, substitutions, such as conservative substitutions) but not A nucleic acid sequence of more than 30, 20 or 10 modifications (eg, substitutions, such as conservative substitutions).

In one embodiment, the invention features an isolated nucleic acid molecule encoding an anti-BCMA binding domain, wherein the anti-BCMA binding domain comprises one or more of the anti-BCMA binding domains described herein ( For example, all three) light chain complementarity determining region 1 (LC CDR1), light chain complementarity determining region 2 (LC CDR2) and/or light chain complementarity determining region 3 (LC CDR3), and the anti-BCMA binding structure described herein One or more (eg, all three) heavy chain complementarity determining region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2), and/or heavy chain complementarity determining region 3 (HC CDR3), for example, comprising Or a plurality, for example, all three LC CDRs and one or more, such as the human anti-BCMA binding domain of all three HC CDRs. In one embodiment, the encoded anti-BCMA binding domain comprises a light chain variable region as described herein (eg, SEQ ID NO: 84, 85, 86, 87, 88, 89, 90, 91, 92, 93 , 94, 95, 96, 97, 98, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211 , 212, 259, 260, 261 or 262) and/or heavy chain variable regions as described herein (eg, SEQ ID NO: 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 81, 82, 83, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 255, 256, 257 or 258). In one embodiment, the encoded anti-BCMA binding domain comprises SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO :44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52 SEQ ID NO: 53, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 265 Or the amino acid sequence of SEQ ID NO: 266 or the scFv of the light and heavy chains of the sequence having 95% to 99% identity thereto. In one embodiment, the anti-BCMA binding domain (eg, scFv) comprises: comprising pairs SEQ ID NO: 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96 , 97, 98, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 259, 260 At least one, two or three modifications (eg, substitutions, eg, conservative substitutions) of the amino acid sequence of the light chain variable region provided in 261 or 262 but no more than 30, 20 or 10 modifications (eg, Substituting, for example, conservatively substituted amino acid sequences or with SEQ ID NOs: 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 192, 193 Amino acid of 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 259, 260, 261 or 262 a sequence of light chain variable regions having a sequence of 95%-99% identity; and/or comprising having SEQ ID NOs: 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 81, 82, 83, 171, 172, 173, 174, 175, 176, 177, 178, 179, 1 At least one, two of the amino acid sequences of the heavy chain variable region provided in 80, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 255, 256, 257 or 258 Amino acid sequence of one or three modifications (eg, substitutions, such as conservative substitutions) but no more than 30, 20 or 10 modifications (eg, substitutions, eg, conservative substitutions) or with SEQ ID NOs: 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 81, 82, 83, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, The amino acid sequence of 185, 186, 187, 188, 189, 190, 191, 255, 256, 257 or 258 has a heavy chain variable region of 95% to 99% identity sequence. In one embodiment, the anti-BCMA binding domain comprises selected from the group consisting of SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44 SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO : 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144 SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148 and SEQ ID NO: 149, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 265 or SEQ ID NO: A sequence of a population consisting of 266, or a sequence with 95%-99% identity. In one embodiment, the encoded anti-BCMA binding domain is a scFv and comprises a light chain variable region of a amino acid sequence described herein (eg, in Table 1 or 16) via a linker (eg, as described herein) The linker) is attached to a heavy chain variable region comprising an amino acid sequence as described herein (e.g., in Tables 1 or 16). In one embodiment, the encoded anti-BCMA binding domain comprises a (Gly ₄ -Ser) n linker, wherein n is 1, 2, 3, 4, 5 or 6, preferably 4 (SEQ ID NO: 26). The light chain variable region and the heavy chain variable region of the scFv can, for example, be in any of the following orientations: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain Variable zone. In one embodiment, the isolated nucleic acid sequence encoding a human anti-BCMA binding domain comprises selected from the group consisting of SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58. SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164. The sequence of the population consisting of SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, and SEQ ID NO: 170, or 95%-99 thereof The sequence of % consistency.

In other embodiments, the encoded BCMA binding domain comprises the HC CDR1, HC CDR2, and HC CDR3 of any of the BCMA heavy chain binding domain amino acid sequences set forth in Tables 1 or 16. In an embodiment, the BCMA binding domain further LC CDR1, LC CDR2 and LC CDR3. In an embodiment, the BCMA binding domain comprises the LC CDR1, LC CDR2 and LC CDR3 of any of the BCMA light chain binding domain amino acid sequences listed in Table 1 or 16.

In some embodiments, the encoded BCMA binding domain comprises one of LC CDR1, LC CDR2 and LC CDR3 of any of the BCMA light chain binding domain amino acid sequences listed in Table 1 or 16 or both All, and one, both or all of the HC CDR1, HC CDR2 and HC CDR3 of any of the BCMA heavy chain binding domain amino acid sequences listed in Table 1 or 16.

In one embodiment, the anti-BCMA binding domain (eg, scFv) comprises: an amino acid sequence comprising a light chain variable region comprising (or consisting of) SEQ ID NO: 271 or 273, and having the pair of SEQ ID NO: 271 or 273, at least one, two or three modifications (eg, substitutions, such as conservative substitutions) of the amino acid sequence of the light chain variable region but no more than 30, 20 or 10 modifications ( For example, a substituted, for example conservatively substituted, amino acid sequence, or a light chain variable region thereof having a sequence with 95%-99% identity; and/or comprising (or consisting of) SEQ ID NO: 271 or 273 An amino acid sequence of the heavy chain variable region, and at least one, two or three modifications (eg, substitutions) of the amino acid sequence of the heavy chain variable region provided in SEQ ID NO: 271 or 273 For example, a conservative substitution) but no more than 30, 20 or 10 modifications (e.g., substitutions, such as conservative substitutions) of the amino acid sequence, or a heavy chain variable region thereof having 95% to 99% identity. In one embodiment, the encoded humanized anti-BCMA binding domain is a scFv and comprises a light chain variable region as described herein, eg, the amino acid sequence provided in SEQ ID NO: 271 or 273, via ligation A linker, such as a linker described herein, is attached to a heavy chain variable region comprising an amino acid sequence as described herein, such as set forth in SEQ ID NO: 271 or 273. In one embodiment, the encoded anti-BCMA binding domain comprises a (Gly ₄ -Ser) n linker, wherein n is 1, 2, 3, 4, 5 or 6, preferably 4 (SEQ ID NO: 26).

In another aspect, the invention relates to an isolated polypeptide molecule, such as an isolated chimeric antigen receptor (CAR) molecule, encoded by the nucleic acid molecule. In one embodiment, the isolated polypeptide molecule comprises a molecule selected from the group consisting of SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO : 113, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220 SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ The sequence of the group consisting of ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, and SEQ ID NO: 233, or a sequence having 95% to 99% identity thereto.

In another aspect, the invention relates to an isolated chimeric antigen receptor (CAR) a molecule (eg, a polypeptide) comprising an anti-BCMA binding domain (eg, a human or humanized antibody or antibody fragment that specifically binds to BCMA), a transmembrane domain, and an intracellular signaling domain (eg, comprising a costimulatory domain) And/or the intracellular signaling domain of the major signaling domain). In one embodiment, the CAR comprises an anti-BCMA binding domain as described herein (eg, a human antibody or antibody fragment that specifically binds to BCMA as described herein), a transmembrane domain as described herein, and herein An antibody or antibody fragment of an intracellular signaling domain (eg, an intracellular signaling domain comprising a co-stimulatory domain and/or a major signaling domain as described herein).

In one embodiment, the anti-BCMA binding domain comprises one or more (eg, all three) light chain complementarity determining regions 1 (LC CDR1), light chain complementarity determining regions of the anti-BCMA binding domain described herein. 2 (LC CDR2) and light chain complementarity determining region 3 (LC CDR3), and one or more (eg, all three) heavy chain complementarity determining regions 1 (HC CDR1) of the anti-BCMA binding domain described herein, Heavy chain complementarity determining region 2 (HC CDR2) and heavy chain complementarity determining region 3 (HC CDR3), eg, human or comprising one or more, eg, all three, LC CDRs and one or more, eg, all three HC CDRs Humanized anti-BCMA binding domain. In one embodiment, the anti-BCMA binding domain comprises a light chain variable region as described herein (eg, in Table 1 or SEQ ID NO: 271 or 273) and/or a heavy chain variable region as described herein (eg, Table 1 or SEQ ID NO: 271 or 273). In one embodiment, the anti-BCMA binding domain is a scFv comprising the light and heavy chains of the amino acid sequence set forth in Table 1, SEQ ID NO: 271 or 273. In one embodiment, the anti-BCMA binding domain (eg, scFv) comprises: at least one, two comprising an amino acid sequence having a light chain variable region as provided in Table 1 or SEQ ID NO: 271 or 273 One or three modifications (eg, substitutions, such as conservative substitutions) but no more than 30, 20 or 10 modifications (eg, substitutions, such as conservative substitutions) of the amino acid sequence, or with Table 1 or SEQ ID NO: 271 or The amino acid sequence provided in 273 has a light chain variable region of 95%-99% identity; and/or comprises a heavy chain variable provided in Table 1 or SEQ ID NO: 271 or 273 At least one, two or three modifications (eg, substitutions, such as conservative substitutions) of the amino acid sequence of the region but no more than 30, 20 or 10 modifications (eg, substitutions, such as conservative substitutions) of the amino acid sequence, Or a heavy chain variable region of a sequence having 95% to 99% identity with the amino acid sequence provided in Table 1 or SEQ ID NO: 271 or 273. In one embodiment, the anti-BCMA binding domain comprises selected from the group consisting of SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44 SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO : 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144 SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 265 or SEQ ID NO: a sequence of a population consisting of 266; or having at least one, two or three modifications (eg, substitutions, eg, conservative substitutions) to any of the foregoing sequences, but no more than 30, 20, or 10 modifications (eg, substitutions) , for example, a conservatively substituted amino acid sequence; or with either Said sequence having 95% sequence identity of 99%. In one embodiment, the anti-BCMA binding domain comprises a sequence selected from the group consisting of SEQ ID NO: 271 or SEQ ID NO: 273 or a sequence having 95% to 99% identity thereto. In one embodiment, the anti-BCMA binding domain is a scFv and comprises a light chain variable region as described herein, eg, the amino acid sequence of Table 1 or 16, SEQ ID NO:271 or SEQ ID NO:273 Attachment to a heavy chain variable region comprising an amino acid sequence as described herein, such as Table 1 or 16, SEQ ID NO: 271 or SEQ ID NO: 273, via a linker, such as a linker as described herein. In one embodiment, the anti-BCMA binding domain comprises a (Gly ₄ -Ser) n linker, wherein n is 1, 2, 3, 4, 5 or 6, preferably 4 (SEQ ID NO: 26). The light chain variable region and the heavy chain variable region of the scFv can, for example, be in any of the following orientations: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain Variable zone.

In other embodiments, the BCMA binding domain comprises the HC CDR1, HC CDR2 and HC CDR3 of any of the BCMA heavy chain binding domain amino acid sequences listed in Table 1 or 16. In an embodiment, the BCMA binding domain further comprises LC CDR1, LC CDR2, and LC CDR3. In an embodiment, the BCMA binding domain comprises the LC CDR1, LC CDR2 and LC CDR3 of any of the BCMA light chain binding domain amino acid sequences listed in Table 1 or 16.

In some embodiments, the BCMA binding domain comprises one, two or all of LC CDR1, LC CDR2 and LC CDR3 of any BCMA light chain binding domain amino acid sequence set forth in Table 1 or 16, and One, both or all of the HC CDR1, HC CDR2 and HC CDR3 of any of the BCMA heavy chain binding domain amino acid sequences listed in Table 1 or 16.

In one embodiment, the isolated CAR molecule comprises, for example, a transmembrane domain of a protein selected from the group consisting of: alpha, beta or ξ chain of a T cell receptor, CD28, CD3 epsilon, CD45, eg, as described herein. , CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154. In one embodiment, the transmembrane domain comprises the sequence of SEQ ID NO: 6. In one embodiment, the transmembrane domain comprises at least one, two or three modifications (eg, substitutions, eg, conservative substitutions) to the amino acid sequence of SEQ ID NO: 6, but no more than 20, 10 or The amino acid sequence of 5 modifications (e.g., substitutions, such as conservative substitutions), or sequences having 95% to 99% identity to the amino acid sequence of SEQ ID NO: 6.

In one embodiment, the anti-BCMA binding domain is joined to the transmembrane domain by a hinge region, such as the hinge region described herein. In one embodiment, the encoded hinge region comprises SEQ ID NO: 2 or a sequence with 95%-99% identity thereto.

In one embodiment, the isolated CAR molecule further comprises a coded co-stimulatory structure The sequence of a domain, such as the costimulatory domain described herein. In an embodiment, the intracellular signaling domain of the isolated CAR molecule comprises a costimulatory domain. In an embodiment, the intracellular signaling domain of the isolated CAR molecule comprises a major signaling domain. In an embodiment, the intracellular signaling domain of the isolated CAR molecule comprises a costimulatory domain and a major signaling domain.

In one embodiment, the costimulatory domain comprises a functional signaling domain of a protein selected from the group consisting of: MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, Integrin, signaling lymphocyte activating molecule (SLAM protein), activated NK cell receptor, BTLA, Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA -1 (CD11a/CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 ( NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a and specific binding to CD83 Body.

In one embodiment, the costimulatory domain comprises the sequence of SEQ ID NO:7. In one embodiment, the costimulatory domain comprises an amino acid sequence having SEQ ID NO: At least one, two or three modifications of the column (eg, substitutions, eg, conservative substitutions) but no more than 20, 10 or 5 modifications (eg, substitutions, eg, conservative substitutions) of the amino acid sequence or with SEQ ID NO: The amino acid sequence of 7 has a sequence of 95%-99% identity. In another embodiment, the co-stimulatory domain of CD28 comprises the amino acid sequence of SEQ ID NO: 1104. In one embodiment, the costimulatory domain comprises an amino acid sequence having at least one, two or three modifications but no more than 20, 10 or 5 modifications to the amino acid sequence of SEQ ID NO: 1104 Or a sequence having 95%-99% identity with the amino acid sequence of SEQ ID NO: 1104. In another embodiment, the costimulatory domain of CD27 comprises the amino acid sequence of SEQ ID NO: 8. In one embodiment, the costimulatory domain comprises an amino acid sequence having at least one, two or three modifications to the amino acid sequence of SEQ ID NO: 8, but no more than 20, 10 or 5 modifications. Or a sequence having 95%-99% identity to the amino acid sequence of SEQ ID NO: 8. In another embodiment, the co-stimulatory domain of ICOS comprises the amino acid sequence of SEQ ID NO: 1106. In one embodiment, the costimulatory domain comprises an amino acid sequence having at least one, two or three modifications to the amino acid sequence of SEQ ID NO: 1106 but no more than 20, 10 or 5 modifications Or a sequence having 95%-99% identity with the amino acid sequence of SEQ ID NO: 1106.

In an embodiment, the major signaling domain comprises a signaling domain of CD3ξ. In an embodiment, the functional signaling domain of CD3ξ comprises SEQ ID NO: 9 (mutant CD3ξ) or SEQ ID NO: 10 (wild-type human CD3ξ) or a sequence with 95%-99% identity thereto.

In one embodiment, the intracellular signaling domain comprises a functional signaling domain of 4-1BB and/or a functional signaling domain of CD3ξ. In one embodiment, the intracellular signaling domain comprises the sequence of SEQ ID NO: 7 and/or the sequence of SEQ ID NO: 9 or SEQ ID NO: 10. In one embodiment, the intracellular signaling domain comprises at least one, two having an amino acid sequence of SEQ ID NO: 7 and/or an amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10. Or three modifications (eg substitutions, such as conservation) Substituting) but not more than 20, 10 or 5 modifications (eg, substitutions, such as conservative substitutions) of the amino acid sequence, or with the amino acid sequence of SEQ ID NO: 7 and/or SEQ ID NO: 9 or SEQ ID NO The amino acid sequence of 10 has a sequence of 95%-99% identity. In one embodiment, the intracellular signaling domain comprises the sequence of SEQ ID NO: 7 and/or the sequence of SEQ ID NO: 9 or SEQ ID NO: 10, wherein the sequences comprising the intracellular signaling domain are Expressed in the same frame and in the form of a single polypeptide chain.

In one embodiment, the intracellular signaling domain comprises a functional signaling domain of CD27 and/or a functional signaling domain of CD3ξ. In one embodiment, the intracellular signaling domain of CD27 comprises the amino acid sequence of SEQ ID NO: 8 and/or the CD3 glutamic acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10. In one embodiment, the intracellular signaling domain comprises at least one, two having an amino acid sequence of SEQ ID NO: 8 and/or an amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10. Or three modified but no more than 20, 10 or 5 modified amino acid sequences, or an amino acid sequence of SEQ ID NO: 8 and/or an amine of SEQ ID NO: 9 or SEQ ID NO: 10. The base acid sequence has a sequence of 95%-99% identity. In one embodiment, the intracellular signaling domain comprises the sequence of SEQ ID NO: 8 and the sequence of SEQ ID NO: 9 or SEQ ID NO: 10, wherein the sequences constituting the intracellular signaling domain are in the same frame It is expressed in the form of a single polypeptide chain.

In one embodiment, the intracellular signaling domain comprises a functional signaling domain of CD28 and/or a functional signaling domain of CD3ξ. In one embodiment, the encoded intracellular signaling domain of CD28 comprises the amino acid sequence of SEQ ID NO: 1104 and/or the CD3 glutamic acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10. In one embodiment, the intracellular signaling domain comprises at least one, two having the amino acid sequence of SEQ ID NO: 1104 and/or the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10. Or three modified but no more than 20, 10 or 5 modified amino acid sequences, or with the amino acid sequence of SEQ ID NO: 379 and/or SEQ ID NO: 9 or SEQ ID NO: The amino acid sequence of 10 has a sequence of 95%-99% identity. In one embodiment, the intracellular signaling domain comprises the sequence of SEQ ID NO: 1104 and the sequence of SEQ ID NO: 9 or SEQ ID NO: 10, wherein the sequences constituting the intracellular signaling domain are in the same frame It is expressed in the form of a single polypeptide chain.

In one embodiment, the isolated CAR molecule further comprises a leader sequence, such as the leader sequence described herein. In one embodiment, the leader sequence comprises the amino acid sequence of SEQ ID NO: 1, or a sequence that is 95%-99% identical to the amino acid sequence of SEQ ID NO: 1.

In another aspect, the invention relates to an isolated CAR molecule comprising a leader sequence, such as a leader sequence as described herein, eg, a leader sequence of SEQ ID NO: 1 or having 95%-99% identity thereto; An anti-BCMA binding domain as described herein, eg, an anti-BCMA binding domain comprising LC CDR1, LC CDR2, LC CDR3, HC CDR1, HC CDR2 and HC CDR3 as described herein, eg, Table 1 or 16, SEQ ID NO: 271 or the anti-BCMA binding domain set forth in SEQ ID NO: 273, or a sequence having 95% to 99% identity thereto; a hinge region, such as the hinge region described herein, eg, SEQ ID NO: 2 The hinge region, or 95%-99% identical thereto; a transmembrane domain, such as a transmembrane domain as described herein, for example having the sequence of SEQ ID NO: 6 or having 95%-99% identity thereto A transmembrane domain of a sequence; an intracellular signaling domain, such as an intracellular signaling domain as described herein (eg, an intracellular signaling domain comprising a costimulatory domain and/or a major signaling domain). In one embodiment, the intracellular signaling domain comprises a costimulatory domain, such as a costimulatory domain as described herein, eg, having the sequence of SEQ ID NO: 7 or having a 95%-99% identity thereto a 1BB co-stimulatory domain; and/or a major signaling domain, such as a major signaling domain as described herein, eg, having the sequence of SEQ ID NO: 9 or SEQ ID NO: 10 or consistent with 95%-99% thereof Sexual CD3ξ stimulation domain. In one embodiment, the intracellular signaling domain comprises a costimulatory domain, such as the co-stimulation described herein a domain (eg, a 4-1BB co-stimulatory domain having the sequence of SEQ ID NO: 7); and/or a major signaling domain, such as the major signaling domain described herein, for example, having SEQ ID NO: 9 or CD3ξ stimulation domain of the sequence of SEQ ID NO: 10.

In one embodiment, the isolated CAR molecule comprises (eg, consists of) SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO :104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112 SEQ ID NO: 113, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO : 228, the amino acid sequence of SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232 or SEQ ID NO: 233, or having SEQ ID NO: 99, SEQ ID NO :100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108 SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, S EQ ID NO: 113, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, at least one, two, three, four, five, ten of SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232 or SEQ ID NO: 15, 20 or 30 modifications (eg, substitutions, such as conservative substitutions) but no more than 60, 50 or 40 modifications (eg, substitutions, such as conservative substitutions) of amino acid sequences, Or with SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, Amino acid sequence of SEQ ID NO: 231, SEQ ID NO: 232 or SEQ ID NO: 233 having an amino acid sequence of 85%, 90%, 95%, 96%, 97%, 98% or 99% identity .

In other embodiments, the anti-BCMA binding domain comprises the HC CDR1, HC CDR2 and HC CDR3 of any of the BCMA heavy chain binding domain amino acid sequences listed in Table 1 or 16. In an embodiment, the BCMA binding domain further comprises LC CDR1, LC CDR2, and LC CDR3. In an embodiment, the BCMA binding domain comprises the LC CDR1, LC CDR2 and LC CDR3 of any of the BCMA light chain binding domain amino acid sequences listed in Table 1 or 16.

In some embodiments, the anti-BCMA binding domain comprises one, both or all of LC CDR1, LC CDR2 and LC CDR3 of any BCMA light chain binding domain amino acid sequence set forth in Table 1 or And one, both or all of the HC CDR1, HC CDR2 and HC CDR3 of any of the BCMA heavy chain binding domain amino acid sequences listed in Tables 1 or 16.

In one aspect, the invention relates to a BCMA binding domain comprising one or more (eg, all three) of the BCMA binding domains described herein, a light chain complementarity determining region 1 (LC CDR1), a light chain Complementarity determining region 2 (LC CDR2) and light chain complementarity determining region 3 (LC CDR3), and/or one or more of the BCMA binding domains described herein (eg, full 3) heavy chain complementarity determining region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2) and heavy chain complementarity determining region 3 (HC CDR3), for example comprising one or more, for example all three LC CDRs And one or more, such as the BCMA binding domains of all three HC CDRs.

In other embodiments, the BCMA binding domain comprises the HC CDR1, HC CDR2 and HC CDR3 of any of the BCMA heavy chain binding domain amino acid sequences listed in Table 1 or 16. In an embodiment, the BCMA binding domain further comprises LC CDR1, LC CDR2, and LC CDR3. In an embodiment, the BCMA binding domain comprises the LC CDR1, LC CDR2 and LC CDR3 of any of the BCMA light chain binding domain amino acid sequences listed in Table 1 or 16.

In some embodiments, the BCMA binding domain comprises one, two or all of LC CDR1, LC CDR2 and LC CDR3 of any BCMA light chain binding domain amino acid sequence set forth in Table 1 or 16, and One, both or all of the HC CDR1, HC CDR2 and HC CDR3 of any of the BCMA heavy chain binding domain amino acid sequences listed in Table 1 or 16.

In one embodiment, the BCMA binding domain comprises one or more (eg, all three) of the BCMA binding domains described herein, a light chain complementarity determining region 1 (LC CDR1), a light chain complementarity determining region 2 (LC CDR2) And light chain complementarity determining region 3 (LC CDR3), and one or more (eg, all three) heavy chain complementarity determining regions 1 (HC CDR1), heavy chain complementarity determining region 2 of the BCMA binding domain described herein (HC CDR2) and heavy chain complementarity determining region 3 (HC CDR3), for example, human or humanized anti-BCMA binding comprising one or more, for example all three, LC CDRs and one or more, for example all three HC CDRs Domain. In one embodiment, the BCMA binding domain comprises a light chain variable region as described herein (eg, in Table 1 or SEQ ID NO: 271 or 273) and/or a heavy chain variable region as described herein (eg, Table 1) Or SEQ ID NO: 271 or 273). In one embodiment, the BCMA binding domain is a scFv comprising the light and heavy chains of the amino acid sequences set forth in Table 1, SEQ ID NO: 271 or 273. In one embodiment, the BCMA binding domain (eg, scFv) comprises: at least one, two or comprising an amino acid sequence having a light chain variable region as provided in Table 1 or SEQ ID NO: 271 or 273 An amino acid sequence of three modifications (eg, substitutions, eg, conservative substitutions) but no more than 30, 20 or 10 modifications (eg, substitutions, eg, conservative substitutions), or with Table 1 or SEQ ID NO: 271 or 273 The amino acid sequence provided has a light chain variable region of 95%-99% identity; and/or comprises a heavy chain variable region as provided in Table 1 or SEQ ID NO: 271 or 273 An amino acid sequence of at least one, two or three modifications (eg, substitutions, eg, conservative substitutions) of the amino acid sequence but no more than 30, 20 or 10 modifications (eg, substitutions, eg, conservative substitutions), or The amino acid sequence provided in Table 1 or SEQ ID NO: 271 or 273 has a heavy chain variable region of 95% to 99% identity. In one embodiment, the BCMA binding domain comprises selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO :53, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136 SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 265 or SEQ ID NO a sequence of 266 consisting of; or having at least one, two or three modifications (eg, substitutions, such as conservative substitutions) to any of the foregoing sequences, but no more than 30, 20 or 10 modifications (eg, substitutions, eg, A conservatively substituted amino acid sequence; or with either 95% -99% sequence having a sequence identity of. In one embodiment, the BCMA binding domain comprises a sequence selected from the group consisting of SEQ ID NO: 271 or SEQ ID NO: 273 or a sequence having 95%-99% identity thereto. In one embodiment, the anti-BCMA binding domain is a scFv and comprises a light chain variable region as described herein, eg, the amino acid sequence of Table 1 or 16, SEQ ID NO:271 or SEQ ID NO:273 Attachment to a heavy chain variable region comprising an amino acid sequence as described herein, such as Table 1 or 16, SEQ ID NO: 271 or SEQ ID NO: 273, via a linker, such as a linker as described herein. In one embodiment, the BCMA binding domain comprises a (Gly ₄ -Ser) n linker, wherein n is 1, 2, 3, 4, 5 or 6, preferably 4 (SEQ ID NO: 26). The light chain variable region and the heavy chain variable region of the scFv can, for example, be in any of the following orientations: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain Variable zone.

In another aspect, the invention features a vector comprising a nucleic acid molecule described herein, such as a nucleic acid molecule encoding a CAR as described herein. In one embodiment, the vector is selected from the group consisting of DNA, RNA, plastid, lentiviral vector, adenoviral vector or retroviral vector.

In one embodiment, the vector is a lentiviral vector. In one embodiment, the vector further comprises a promoter. In one embodiment, the promoter is an EF-1 promoter. In one embodiment, the EF-1 promoter comprises the sequence of SEQ ID NO:11. In another embodiment, the promoter is a PGK promoter, such as a truncated PGK promoter as described herein.

In one embodiment, the vector is an ex vivo transcription vector, such as a vector that transcribes RNA of a nucleic acid molecule described herein. In one embodiment, the nucleic acid sequence in the vector further comprises a poly(A) tail, such as the poly A tail described herein, for example comprising about 150 adenosine bases (SEQ ID NO: 382). In one embodiment, the nucleic acid sequence in the vector further comprises a 3 'UTR, such as a 3 'UTR as described herein, for example comprising at least one repeat sequence derived from the 3 'UTR of human β-globulin. In one embodiment, the nucleic acid sequence in the vector further comprises a promoter, such as a T2A promoter.

In another aspect, the invention relates to a cell comprising a vector described herein. In one embodiment, the cell is a cell described herein, such as an immune effector cell, such as a human T cell or a human NK cell, such as a human T cell or a human described herein. Human NK cells as described herein. In one embodiment, the human T cell is a CD8+ T cell.

In another embodiment, the CAR-expressing cells described herein can further express another agent, such as an agent that increases the activity of the cells expressing the CAR. For example, in one embodiment, the agent can be an agent that inhibits the inhibitory molecule. Examples of inhibitory molecules include PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (eg, CEACAM-1, CEACAM-3, and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine and TGFR beta. In an embodiment, the agent is an agent that inhibits PD1. In an embodiment, the agent is an agent that inhibits PD-L1. In one embodiment, the agent that inhibits the inhibitory molecule can be an agent described herein, such as a first polypeptide (eg, an inhibitory molecule) and a positive signal to a cell, such as the intracellular signaling domain described herein. A second polypeptide-associated agent. In one embodiment, the agent comprises a first polypeptide, such as an inhibitory molecule, such as PD1, PD-L1, PD-L2, LAG3, CEACAM (eg, CEACAM-1, CEACAM-3, and/or CEACAM-5), CTLA4, VISTA, CD160, BTLA, LAIR1, TIM3, 2B4, TGFR β, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC II a class, GAL9, adenosine, and TIGIT, or a fragment of any of these (eg, at least a portion of an extracellular domain of any of these); and a second polypeptide that is intracellular as described herein A signaling domain (eg, comprising a costimulatory domain (eg, 41BB, CD27, or CD28, eg, as described herein) and/or a primary signaling domain (eg, a CD3ξ signaling domain as described herein). In one embodiment The agent comprises a first polypeptide of PD1 or a fragment thereof (eg, at least a portion of the extracellular domain of PD1), and an intracellular signaling domain as described herein (eg, a CD28 signaling domain as described herein and/or herein) a second polypeptide of the CD3ξ signaling domain).

In another aspect, the invention relates to a method of producing a cell comprising transducing a cell described herein, such as an immune effector cell described herein, with a vector comprising a nucleic acid encoding a CAR, such as a CAR as described herein. For example, T cells or NK cells as described herein.

The invention also provides a method of producing a population of RNA engineered cells, such as cells described herein, such as immune effector cells, such as T cells or NK cells, which transiently exhibit exogenous RNA. The method comprises introducing an in vitro transcribed RNA or synthetic RNA into a cell, wherein the RNA comprises a nucleic acid encoding a CAR molecule as described herein.

In another aspect, the invention relates to a method of providing anti-tumor immunity in a mammal comprising administering to the mammal an effective amount of a cell expressing a CAR molecule, such as a cell expressing a CAR molecule as described herein . In one embodiment, the cell is an autoimmune effector cell, such as a T cell or an autologous NK cell. In one embodiment, the cells are immune effector cells, such as allogeneic T cells or allogeneic NK cells. In one embodiment, the mammal is a human, such as a patient with hematological cancer.

In another aspect, the invention relates to a method of treating a mammal having a disease associated with BCMA performance, such as a proliferative disease associated with BCMA performance, a precancerous condition, and a non-cancer related indication, A cell comprising a potent amount of a CAR molecule, such as a CAR molecule as described herein, is administered to the mammal. In one embodiment, the mammal is a human, such as a patient with hematological cancer.

In one embodiment, the disease is a disease as described herein. In one embodiment, the disease associated with BCMA performance is selected from the group consisting of a blood cancer, such as acute leukemia, including but not limited to acute myeloid leukemia (AML); myelodysplastic syndrome; myeloablative neoplasms; chronic myeloid leukemia (CML); parental plasmacytoid dendritic cell neoplasms; and diseases associated with BCMA manifestations, including but not limited to atypical and/or non-classical cancers, malignant diseases, precancerous conditions showing BCMA Or a proliferative disease; and combinations thereof. In one embodiment, the disease associated with BCMA performance is selected from the following Blood cancer consisting of one or more acute leukemias including, but not limited to, B-cell acute lymphoblastic leukemia ("BALL"), T-cell acute lymphocytic leukemia ("TALL"), acute lymphocytic leukemia (ALL) ; one or more chronic leukemias, including but not limited to chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL); other hematological or hematological conditions, including but not limited to B cells Lymphocytic leukemia, parental plasmacytoid dendritic cell tumor, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cells or Large cell follicular lymphoma, malignant lymphoproliferative condition, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma , plasmablastic lymphoma, plasmacytoid dendritic cell tumor, Waldenstrom macroglobulinemia and "leukemia prodrosis", leukemia prodrosis is composed of bone Blood cell ineffective (or dysplastic) combined with various blood conditions; and diseases associated with BCMA performance, including but not limited to atypical and/or non-classical cancers, malignant diseases, precancerous conditions or hyperplasia of BCMA Sexual diseases; and combinations thereof.

In the examples, diseases associated with BCMA manifestations include plasma cell proliferative disorders such as asymptomatic myeloma (and suspicion of multiple myeloma or inert myeloma), unexplained gamma globulinemia (MGUS), var. Denver's macroglobulinemia, plasmacytoma (eg plasma cell cachexia, solitary myeloma, solitary plasmacytoma, extramedullary plasmacytoma and multiple plasmacytoma), systemic amyloid light chain Amyloidosis and POEMS syndrome (also known as Crow-Fukase syndrome, Takatsuki disease, and PEP syndrome).

In an embodiment, the disease associated with BCMA performance includes cancer, such as a cancer as described herein, such as prostate cancer (eg, castration resistant or therapeutic resistant prostate cancer, or metastatic prostate cancer), pancreatic cancer, or lung cancer.

In one embodiment of the method of treatment, the CAR molecules described herein are expressed (eg, Cells of the BCMA CAR molecule are administered in combination with cells containing the CD19 CAR molecule. In one embodiment, cells expressing BCMA CAR molecules are administered before, after, or at the same time as cells expressing CD19 CAR are administered. In one embodiment, the cells expressing the BCMA CAR molecule and the cells expressing the CD19 CAR molecule are part of a single composition, and in other embodiments, the cells expressing the BCMA CAR molecule and the cells expressing the CD19 CAR molecule are separate compositions. Part of it. In one embodiment, cells expressing a CAR molecule (eg, a BCMA CAR molecule) described herein also exhibit a CD19 CAR molecule. In one embodiment, the disease associated with BCMA is multiple myeloma, such as CD19-negative multiple myeloma. In one embodiment, the disease associated with BCMA performance is multiple myeloma, such as CD19 negative, such as by flow cytometry and RT-PCR detection, for example, having a majority (eg, 99.95%) Multiple myeloma of neoplastic plasma cells with a CD19 negative phenotype.

In one embodiment, a cell that expresses a CAR molecule, such as a CAR molecule described herein, is administered in combination with an agent that increases the efficacy of a cell that exhibits a CAR molecule, such as an agent described herein.

In one embodiment, cells expressing a CAR molecule (eg, a CAR molecule described herein) are administered in combination with a low immunopotentiating dose of the mTOR inhibitor. Although not wishing to be bound by theory, a low-immunization-enhancing dose of salty credit (eg, a dose sufficient to completely suppress the immune system but sufficient to increase immune function) is accompanied by a decrease in PD-1 positive immune effector cells, such as T cells or NK cells, or PD-1 negative cells increased. PD-1 positive immune effector cells (eg, T cells or NK cells) can be depleted by binding to cells expressing PD-1 ligands, such as PD-L1 or PD-L2, rather than PD-1 negative immune effects Cells (eg T cells or NK cells).

In one embodiment, this method can be used to optimize the efficacy of the CAR cells described herein in an individual. While not wishing to be bound by theory, it is believed that in one embodiment, the efficacy of endogenous, unmodified immune effector cells, such as T cells or NK cells, is enhanced. although Without wishing to be bound by theory, it is believed that in one embodiment, the performance of cells expressing BCMA CAR is enhanced. In other embodiments, cells that have been or will be engineered to express CAR, such as immune effector cells (eg, T cells or NK cells), can increase PD1 negative immune effector cells (eg, T cells or NK cells) by exposure. The amount or amount of mTOR inhibitor that increases the ratio of PD1 negative immune effector cells (eg, T cells or NK cells) / PD1 positive immune effector cells (eg, T cells or NK cells) is treated ex vivo.

In one embodiment, administration of a low immunopotentiating dose of an mTOR inhibitor, such as an allosteric inhibitor, for example, prior to administration of a cell expressing CAR as described herein, such as an immune effector cell (eg, a T cell or NK cell), eg, RAD001. In one embodiment, after sufficient time or sufficient to administer an mTOR inhibitor, the CAR cells are administered such that PD1 negative immune effector cells (eg, T cells or NK cells) or PD1 negative immune effector cells (eg, T cells or The ratio of NK cells)/PD1 positive immune effector cells (such as T cells or NK cells) increases at least temporarily.

In one embodiment, cells to be engineered to express CAR, such as immune effector cells (eg, T cells or NK cells), are harvested after a sufficient time to administer a low immunopotentiating dose of the mTOR inhibitor or after a sufficient dose is administered The content of PD1 negative immune effector cells (such as T cells or NK cells) or PD1 negative immune effector cells (such as T cells or NK cells) / PD1 positive immune effector cells (such as T cells or NK) in individuals or harvested from individuals. The ratio of cells) increases at least temporarily.

In one embodiment, the invention provides an mTOR inhibitor for treating an individual, wherein the mTOR inhibitor enhances an immune response in the individual, and wherein the individual has accepted, is receiving, or is about to receive a BCMA as described herein CAR immune effector cells. In one embodiment, a cell that exhibits a CAR molecule, such as a CAR molecule described herein, is administered in combination with an agent that ameliorates one or more side effects associated with the administration of a cell that exhibits a CAR molecule, such as the agents described herein.

In one embodiment, a CAR molecule is expressed, such as a fine CAR molecule as described herein. The cells are administered in combination with an agent that treats a disease associated with BCMA, such as an agent described herein.

In certain embodiments, the disease associated with BCMA is a proliferative disease, such as a cancer or malignant disease or precancerous condition, such as myelodysplasia, myelodysplastic syndrome, or leukemia prodromal, or is associated with BCMA performance. Cancer related indications.

In certain embodiments, the disease associated with BCMA is a blood cancer selected from the group consisting of: one or more acute leukemias including, but not limited to, acute myeloid leukemia (AML); myelodysplastic syndrome; Myeloproliferative neoplasms; chronic myeloid leukemia (CML); parental plasmacytoid dendritic cell tumor; multiple myeloma; and diseases associated with BCMA performance, including but not limited to atypical BCMA And/or non-classical cancer, malignant disease, precancerous condition or proliferative disease; and combinations thereof. In one embodiment, the disease associated with BCMA is multiple myeloma. In the examples, diseases associated with BCMA manifestations include plasma cell proliferative disorders such as asymptomatic myeloma (and suspicion of multiple myeloma or inert myeloma), unexplained gamma globulinemia (MGUS), var. Denver's macroglobulinemia, plasmacytoma (eg plasma cell cachexia, solitary myeloma, solitary plasmacytoma, extramedullary plasmacytoma and multiple plasmacytoma), systemic amyloid light chain Amyloidosis and POEMS syndrome (also known as Crohn-Fox syndrome, sorghum and PEP syndrome). In an embodiment, the disease associated with BCMA performance includes cancer, such as a cancer as described herein, such as prostate cancer (eg, castration resistant or therapeutic resistant prostate cancer, or metastatic prostate cancer), pancreatic cancer, or lung cancer.

In the examples, cells expressing BCMA CAR, such as the cells expressing BCMA CAR described herein, are used to treat individuals with multiple myeloma. In an embodiment, a cell that exhibits BCMA CAR, such as a cell expressing BCMA CAR as described herein, is used to treat an individual having a plasma cell proliferative disorder, such as asymptomatic myeloma (and suspicion of multiple myeloma or Indolent myeloma), unexplained single gamma globulinemia (MGUS), Waldenstrom macroglobulinemia, plasmacytoma (eg plasma cell cachexia, solitary myeloma, solitary plasmacytoma, extramedullary plasmacytoma and multiple plasmacytoma), systemic Amyloid light chain amyloidosis and POEMS syndrome (also known as Crohn-Fox syndrome, sorghum and PEP syndrome). In an embodiment, a cell that exhibits BCMA CAR, such as a cell expressing BCMA CAR as described herein, is used to treat an individual having a cancer, such as a cancer described herein, such as a prostate cancer (eg, castration resistance or therapy) Resistant prostate cancer, or metastatic prostate cancer), pancreatic cancer, or lung cancer.

In an embodiment, cells expressing BCMA CAR, such as cells expressing BCMA CAR as described herein, are administered to an individual according to a dosing schedule, the dosing regimen comprising dose totaling of the cells administered to the individual, eg, a partial dose One, two, three or more separate doses. In an embodiment, the first percentage of the total dose is administered on the first day of treatment, on a subsequent treatment day (eg, second day, third day, fourth day, fifth day, sixth day, or seventh day) Or a later number of days) to cast a second percentage of the total dose, and on a subsequent treatment day (eg, third day, fourth day, fifth day, sixth day, seventh day, eighth day, first Nine days, tenth days or later days, depending on the situation, the third percentage of the total dose (eg, the remaining percentage) is administered. For example, 10% of the total cell dose is delivered on the first day, 30% of the total cell dose is delivered on the second day, and the remaining 60% of the total cell dose is delivered on the third day of treatment. For example, the total cell dose includes 1 to 5 x 10 ⁷ or 1 to 5 x 10 ⁸ BCMA-CART cells.

In an embodiment, a lymphocyte depletion therapy (e.g., Cytoxan, e.g., at 1.5 g/m^<2&gt^; ) is administered to an individual prior to administration of the cell expressing CAR. In an embodiment, lymphocyte depletion therapy (eg, Settledas) is not administered to an individual prior to administration of the cell expressing CAR.

In an embodiment, no depletion of lymphocytes administered with chemotherapy, and cast and a 1- to 5 × 10 ⁷ th of the total dose BCMA-CART cells (e.g. by infusion), wherein the first day of treatment administered and the dose of cells 10%, 30% on the 2nd day of treatment, and 60% on the 3rd day of treatment. In another embodiment, no depletion of lymphocytes administered with chemotherapy, and cast and a 1- to 5 × BCMA-CART total dose of 10 ⁸ of cells (e.g. by infusion), wherein the first day of treatment administered to cells 10% of the dose, 30% administered on the second day of treatment, and 60% on the third day of treatment. In the examples, lymphocyte depletion chemotherapy (Settlers, 1.5 g/m ² ) was administered three days prior to administration of BCMA-CART cells, and then 1 to 5×10 ⁷ total BCMAs were administered. - CART cell dose (e.g., by infusion), wherein 10% of the cell dose is administered on the first day of treatment, 30% is administered on the second day of treatment, and 60% is administered on the third day of treatment. In the examples, lymphocyte depletion chemotherapy (Settlers, 1.5 g/m ² ) was administered three days prior to administration of BCMA-CART cells, and then 1 to 5×10 ⁸ total BCMAs were administered. - CART cell dose (e.g., by infusion), wherein 10% of the cell dose is administered on the first day of treatment, 30% is administered on the second day of treatment, and 60% is administered on the third day of treatment.

In another aspect, the invention relates to a method of modulating an individual prior to cell transplantation, comprising administering to the individual an effective amount of a cell comprising a CAR molecule as described herein. In one embodiment, the cells are transplanted into a stem cell transplant. Stem cell transplantation is a hematopoietic stem cell transplant or a bone marrow transplant. In one embodiment, the cells are transplanted as an allogeneic or autologous transplant.

In one embodiment, adjusting the individual prior to cell transplantation comprises reducing the number of cells exhibiting BCMA in the individual. The cells expressing BCMA in an individual are normal cells expressing BCMA or cancer cells expressing BCMA, and in some cases, the condition in the individual will reduce the normality of BCMA and cancer cells prior to cell transplantation.

In another aspect, the invention relates to an isolated nucleic acid molecule encoding a CAR of the invention, an isolated polypeptide molecule of the CAR of the invention, a vector comprising the CAR of the invention, and a cell comprising the CAR of the invention, for example Used as a medicament as described herein.

In another aspect, the invention relates to an isolated nucleic acid molecule encoding a CAR of the invention, an isolated polypeptide molecule of the CAR of the invention, a vector comprising the CAR of the invention, and a cell comprising the CAR of the invention, For the treatment of a disease manifesting BCMA, such as a disease manifesting BCMA as described herein.

Additional features and embodiments of the foregoing compositions and methods include one or more of the following: In certain embodiments, a BCMA CAR molecule (eg, a BCMA CAR nucleic acid or BCMA CAR polypeptide as described herein) or as described herein The BCMA binding domain comprises one, two or three CDRs (eg, HC CDR1, HC CDR2 and/or HC CDR3) from the heavy chain variable region provided in Table 20; and/or provided in Table 21 From BCMA-1, BCMA-2, BCMA-3, BCMA-4, BCMA-5, BCMA-6, BCMA-7, BCMA-8, BCMA-9, BCMA-10, BCMA-11, BCMA-12, BCMA -13, BCMA-14, BCMA-15, 149362, 149363, 149364, 149365, 149366, 149367, 149368, 149369, BCMA_EBB-C1978-A4, BCMA_EBB-C1978-G1, BCMA_EBB-C1979-C1, BCMA_EBB-C1978-C7 , BCMA_EBB-C1978-D10, BCMA_EBB-C1979-C12, BCMA_EBB-C1980-G4, BCMA_EBB-C1980-D2, BCMA_EBB-C1978-A10, BCMA_EBB-C1978-D4, BCMA_EBB-C1980-A2, BCMA_EBB-C1981-C3, BCMA_EBB - one, two or three CDRs of the light chain variable region of C1978-G4, A7D12.2, C11D5.3, C12A3.2, C13F12.1 (eg, LC CDR1, LC CDR2 and/or LC CDR3); With any of the foregoing sequences Consensus sequence (e.g., 95% -99% identical or at most 3, 2 or 1 amino acid changes, such as substituted (e.g. conservatively substituted)) of the mass.

In certain embodiments, a BCMA CAR molecule (eg, a BCMA CAR nucleic acid or BCMA CAR polypeptide as described herein) or an anti-BCMA antigen binding domain as described herein includes the heavy chain variable provided in Table 22 One, two or three CDRs of the region (eg, HC CDR1, HC CDR2 and/or HC CDR3); and/or from BCMA-1, BCMA-2, BCMA-3, BCMA-4, provided in Table 23, BCMA-5, BCMA-6, BCMA-7, BCMA-8, BCMA-9, BCMA-10, BCMA-11, BCMA-12, BCMA-13, BCMA-14, BCMA-15, 149362, 149363, 149364, 149365, 149366, 149367, 149368, 149369, BCMA_EBB-C1978-A4, BCMA_EBB-C1978-G1, BCMA_EBB-C1979-C1, BCMA_EBB-C1978-C7, BCMA_EBB-C1978-D10, BCMA_EBB-C1979-C12, BCMA_EBB-C1980- G4, BCMA_EBB-C1980-D2, BCMA_EBB-C1978-A10, BCMA_EBB-C1978-D4, BCMA_EBB-C1980-A2, BCMA_EBB-C1981-C3, BCMA_EBB-C1978-G4, A7D12.2, C11D5.3, C12A3.2, One, two or three CDRs of the light chain variable region of C13F12.1 (eg, LC CDR1, LC CDR2 and/or LC CDR3); or substantially identical to any of the foregoing sequences (eg, 95%-99% identical or A sequence of up to 5, 4, 3, 2 or 1 amino acid change, such as a substitution (e.g., conservative substitution)). In certain embodiments, the BCMA CAR molecule or anti-BCMA antigen binding domain comprises one, two or three CDRs (eg, HCDR1, HCDR2 and/or HCDR3) from the heavy chain variable region provided in Table 24. And/or provided in Table 25 from BCMA-1, BCMA-2, BCMA-3, BCMA-4, BCMA-5, BCMA-6, BCMA-7, BCMA-8, BCMA-9, BCMA-10 , BCMA-11, BCMA-12, BCMA-13, BCMA-14, BCMA-15, 149362, 149363, 149364, 149365, 149366, 149367, 149368, 149369, BCMA_EBB-C1978-A4, BCMA_EBB-C1978-G1, BCMA_EBB -C1979-C1, BCMA_EBB-C1978-C7, BCMA_EBB-C1978-D10, BCMA_EBB-C1979-C12, BCMA_EBB-C1980-G4, BCMA_EBB-C1980-D2, BCMA_EBB-C1978-A10, BCMA_EBB-C1978-D4, BCMA_EBB-C1980 One, two or three CDRs of the light chain variable region of -A2, BCMA_EBB-C1981-C3, BCMA_EBB-C1978-G4, A7D12.2, C11D5.3, C12A3.2, C13F12.1 (eg LC CDR1) LC CDR2 and/or LC CDR3); or substantially identical to any of the foregoing sequences (eg, 95%-99% identical or at most 5, 4, 3, 2 or 1 amino acid changes, eg, substitutions (eg, conservative substitutions) The sequence of ).

In certain embodiments, the BCMA CAR molecule or the anti-BCMA antigen binding domain LC CDR1, LC CDR2 comprising (i) any BCMA light chain binding domain amino acid sequence listed in Table 1 or 16, in SEQ ID NO: 271 or 273 or in the LC CDRs of Table 21, 23 or 25 And LC CDR3; and/or (ii) any BCMA heavy chain binding domain amino acid sequence listed in Table 1 or 16, in SEQ ID NO: 271 or 273 or in the HC CDRs of Table 20, 22 or 24 HC CDR1, HC CDR2 and HC CDR3.

In certain embodiments, a BCMA molecule (eg, a BCMA CAR nucleic acid or BCMA CAR polypeptide as described herein) or an anti-BCMA antigen binding domain as described herein comprises: (1) three selected from one of the following: Light chain (LC) CDRs: (i) LC CDR1 of SEQ ID NO: 504 of BCMA-4 CAR (139103), LC CDR2 of SEQ ID NO: 544, and LC CDR3 of SEQ ID NO: 584; (ii) BCMA- 10 CAR (139109) LC CDR1 of SEQ ID NO: 514, LC CDR2 of SEQ ID NO: 554 and LC CDR3 of SEQ ID NO: 594; (iii) SEQ ID NO: 516 of BCMA-13 CAR (139112) LC CDR1, LC CDR2 of SEQ ID NO: 556 and LC CDR3 of SEQ ID NO: 596; or (iv) LC CDR1 of SEQ ID NO: 518 of BCMA-15 CAR (139114), LC CDR2 of SEQ ID NO: 558 And LC CDR3 of SEQ ID NO: 598; and/or (2) three heavy chain (HC) CDRs selected from one of the following: (i) BCMA-4 CAR (139103) SEQ ID NO: 384 of HC CDR1 , HC CDR2 of SEQ ID NO: 424 and HC CDR3 of SEQ ID NO: 464; (ii) HC CDR1 of SEQ ID NO: 394 of BCMA-10 CAR (139109), HC CDR2 of SEQ ID NO: 434 and SEQ ID NO: 474, HC CDR3; (iii) BCMA-13 CAR (139112) SEQ ID NO: 396, HC CDR1, SEQ ID NO: 436, HC CDR2 and SEQ ID N O:476 HC CDR3; or (iv) HC CDR1 of SEQ ID NO: 398 of BCMA-15 (139114), HC CDR2 of SEQ ID NO: 438, and HC CDR3 of SEQ ID NO: 478.

In certain embodiments, a BCMA CAR molecule (eg, a BCMA CAR nucleic acid or BCMA CAR polypeptide as described herein) or an anti-BCMA antigen binding domain as described herein comprises: (1) one selected from one of the following: Light chain (LC) CDRs: (i) LC CDR1 of SEQ ID NO: 744 of BCMA-4 CAR (139103), LC CDR2 of SEQ ID NO: 784, and LC CDR3 of SEQ ID NO: 824; (ii) BCMA -10 CAR (139109), LC CDR1 of SEQ ID NO: 754, LC CDR2 of SEQ ID NO: 794, and LC CDR3 of SEQ ID NO: 834; (iii) SEQ ID NO: 756 of BCMA-13 CAR (139112) LC CDR1, LC CDR2 of SEQ ID NO: 796 and LC CDR3 of SEQ ID NO: 836; or (iv) LC CDR1 of SEQ ID NO: 758 of BCMA-15 CAR (139114), LC of SEQ ID NO: 798 CDR2 and LC CDR3 of SEQ ID NO: 838; and/or (2) three heavy chain (HC) CDRs selected from one of the following: (i) BCMA-4 CAR (139103) SEQ ID NO: 624 HC CDR1, HC CDR2 of SEQ ID NO: 664 and HC CDR3 of SEQ ID NO: 704; (ii) HC CDR1 of SEQ ID NO: 634 of BCMA-10 CAR (139109), HC CDR2 of SEQ ID NO: 674 and SEQ ID NO: 714, HC CDR3; (iii) BCMA-13 CAR (139112) SEQ ID NO: 636, HC CDR1, SEQ ID NO: 676, HC CDR2 and SEQ ID NO: 716 HC CDR3; or (iv) BCMA-15 (139114) SEQ ID NO: 638, HC CDR1, SEQ ID NO: 678, HC CDR2, and SEQ ID NO: 718, HC CDR3.

In certain embodiments, a BCMA CAR molecule (eg, a BCMA CAR nucleic acid or BCMA CAR polypeptide as described herein) or an anti-BCMA antigen binding domain as described herein includes: (1) Three light chain (LC) CDRs selected from one of the following: (i) LC CDR1 of SEQ ID NO: 984 of BCMA-4 CAR (139103), LC CDR2 of SEQ ID NO: 1024, and SEQ ID NO LC CDR3 of 1064; (ii) LC CDR1 of SEQ ID NO: 994 of BCMA-10 CAR (139109), LC CDR2 of SEQ ID NO: 1034, and LC CDR3 of SEQ ID NO: 1074; (iii) BCMA-13 CAR (139112), LC CDR1 of SEQ ID NO: 996, LC CDR2 of SEQ ID NO: 1036, and LC CDR3 of SEQ ID NO: 1076; or (iv) SEQ ID NO: 998 of BCMA-15 CAR (139114) LC CDR1, LC CDR2 of SEQ ID NO: 1038 and LC CDR3 of SEQ ID NO: 1078; and/or (2) three heavy chain (HC) CDRs selected from one of: (i) BCMA-4 CAR ( 139103) HC CDR1 of SEQ ID NO: 864, HC CDR2 of SEQ ID NO: 904 and HC CDR3 of SEQ ID NO: 944; (ii) HC CDR1 of SEQ ID NO: 874 of BCMA-10 CAR (139109) HC CDR2 of SEQ ID NO: 914 and HC CDR3 of SEQ ID NO: 954; (iii) HC CDR1 of SEQ ID NO: 876 of BCMA-13 CAR (139112), HC CDR2 of SEQ ID NO: 916 and SEQ ID NO HC CDR3 of: 956; (iv) HC CDR1 of SEQ ID NO: 878 of BCMA-15 CAR (139114), HC CDR2 of SEQ ID NO: 918, and HC CDR3 of SEQ ID NO: 958.

In certain embodiments, a BCMA CAR molecule (eg, a BCMA CAR nucleic acid or BCMA CAR polypeptide as described herein) or an anti-BCMA antigen binding domain as described herein comprises a humanized scFv of SEQ ID NO:271 or 273 An amino acid sequence or a nucleotide sequence encoding the scFv (SEQ ID NO: 272 or 274), or an antigen binding domain thereof (eg, VH, VL or one or more CDRs thereof).

All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, unless otherwise defined. Although methods and materials similar or equivalent to those of the methods and materials described herein can be used in practice or test Invention, but suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are hereby incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting. Titles, subtitles or numbers or elements marked with letters, such as (a), (b), (i), etc., are presented for ease of reading only. The use of headings or numbers or elements marked with letters in this document does not require steps or elements to be performed in alphabetical order, or steps or elements need to be discrete from each other. Other features, objects, and advantages of the present invention will be apparent from the embodiments and the claims.

Figure 1, comprising Figures 1A and 1 B, is a graphical representation of BCMA expression in myeloma samples as determined by quantitative PCR. The BCMA expression in different myeloma cell lines was determined (Fig. 1A). BCMA performance was compared between normal plasma cells and myeloma patient samples (Fig. 1B).

Figure 2, comprising Figures 2A, 2B, 2C, 2D and 2E, is a series of graphical representations of BCMA expression in multiple myeloma cell lines and major samples by flow cytometry. BCMA was detected on the surface of cell lines U266 (Fig. 2A), H929 (Fig. 2B) and 8226 (Fig. 2C). BCMA was also homogeneously expressed on most of the pure plasma cells in 9 of the 10 analyzed multiple myeloma patients (Figures 2D and 2E).

Figure 3, comprising Figures 3A and 3B, is a series of graphical representations demonstrating the lack of BCMA performance in normal peripheral blood cells and after CD3/CD28 amplification (Figure 3A) and normal bone marrow cells (Figure 3B).

Figure 4, comprising Figures 4A, 4B, 4C, 4D, 4E and 4F, is a series of pictures and figures showing BCMA performance in normal tissue. The tissue staining positive for BCMA in immunohistochemical analysis was lymph node (Fig. 4A) and tonsil (Fig. 4B). Representative tissues that are not stained for BCMA performance (BCMA negative) include lung (Fig. 4C), pancreas (Fig. 4D), and thyroid (Fig. 4E). RNA in situ hybridization analysis was also performed in different tissues (Fig. 4F).

Figure 5 is a humanized mouse anti-BCMA scFv containing pBCMA1 Schematic representation of four CAR constructs of pBCMA2, pBCMA3 and pBCMA4.

Figure 6 is a series of flow cell measurement curves showing the transduction efficiency and performance of BMCA-CAR constructs on T cells. SS1-BBz represents anti-mesothelin CAR, which serves as a negative control.

Figure 7, comprising Figures 7A and 7B, is a two-dimensional representation of antigen-specific cytokine production by BCMA-CART as determined by ELISA assay. IL2 (Fig. 7A) and interferon-gamma (IFNg) (Fig. 7B) production were assessed.

Figure 8, comprising Figures 8A, 8B, 8C and 8D, is a series of graphs demonstrating the cytotoxic activity of BCMA-CART on the indicated myeloma cell lines: performance BCMA (Figure 8A); 8226 (Figure 8B) NCI H929 (Fig. 8C); and K562 of OPM2 (Fig. 8D).

Figure 9, comprising Figures 9A and 9B, is a graph and a series of pictures showing the anti-tumor activity of BCMA-CART in a preclinical multiple myeloma animal model. Figure 9A shows the quantification of mean bioluminescence (represented by photons per second) representing disease burden in all animals. Figure 9B shows a picture of bioluminescence detected in treated mice at 5, 15 and 20 days after treatment.

Figure 10, comprising Figures 10A and 10B, is a series of schematic diagrams of a BCMA CAR construct containing a humanized murine anti-BCMA scFv.

Figure 11 is a series of graphs demonstrating the target-specific activation of the BCMA CAR construct transduced in a reporter cell line by luciferase reporter gene analysis.

Figure 12 is a graph showing flow cytometry of the distribution of CD4+ and CD8+ populations of T cells after CD3/CD28 amplification prior to transduction with the tool BCMA CAR construct.

Figure 13 is a series of graphs showing CART transduction efficiency by flow cytometry analysis and corresponding histograms, by detecting BCMA-Fc antigen after 10 days of transduction.

Figure 14 is after stimulation with the indicated target cells (eg K562, K562 expressing BCMA, KMS11-luc, MM1-S-luc, NCI-H929, KMs26, RPMI 8226 and CD3/CD28 beads) CFSE staining, a series of histograms showing the proliferation of the tool BCMA CART cells.

Figure 15, comprising Figures 15A and 15B, is the number of cells by CART-expressing cells (as measured by flow cytometry) after stimulation with the indicated target cells (Figure 15A) and cells The total number (Fig. 15B) shows two plots of proliferation of the tool BCMA CART cells.

Figure 16 shows BCMA CART killing by means of luciferase assay in response to BCMA-targeted cells KMS11-luciferase cells (left) and MM1-S-luciferase cells (right). Figure.

Figure 17 is a series of graphs showing BCMA CART killing in response to target cells expressing BCMA by CFSE cell kill assay.

Figure 18 is a series of graphs demonstrating the target-specific activation of BCMA CAR containing human anti-BCMA scFv transduced in a reported cell line by luciferase reporter gene analysis.

Figure 19 is a graph showing flow cytometry of the distribution of CD4+ and CD8+ T cell populations after CD3/CD28 amplification and prior to CAR transduction.

Figure 20 is a series of flow cytometry curves and corresponding histograms showing the transduction efficiency by assessing CAR performance on transduced T cells.

Figure 21 is a graph showing the proliferation of BCMA CART cells stimulated by CFSE staining in response to stimulation with the indicated target cells (K562, K562 expressing BCMA, RPMI 8226, KM11-luc and NCI-H929). A series of histograms.

Figure 22, comprising Figures 22A, 22B and 22C, is confirmed to be in response to the indicated target cells (K562, K562 expressing BCMA, RPMI 8226, KMS11) as measured by flow cytometry analysis. -luc and NCI-H929) stimulated a series of maps of BCMA CART cell proliferation. Proliferation of CART cells expressing each of the T cell populations of CD3 (Fig. 22A), CD4 (Fig. 22B), and CD8 (Fig. 22C) was independently analyzed.

Figure 23, comprising Figures 23A, 23B and 23C, is a series of graphs showing the killing of BCMA CART in response to KMS11-luciferase target cells expressing BCMA by luciferase assay. Killing ability of each BCMA CAR structure in each of the figures in Figure 23A Cell %) compared to BCMA-3NP and BCMA-4NP. In Figure 23B, the selected BCMA CAR constructs are compared to each other. In Figure 23C, the effector cells: target ratio is corrected relative to cells expressing CAR. The X axis represents the % of target cells killed; the Y axis represents the effect: target (E:T) ratio.

Figure 24 is a graph showing the control of disease progression in KMS-11-luc human multiple myeloma xenografts in NSG mice treated with BCMA CART. The mean bioluminescence (+/- SEM) of tumor cells displays the disease burden in all animals, as shown in the figure as photons per second (p/s) of ROI (relevant regions, eg, all mice). The significance relative to the vehicle was calculated by ANOVA; * indicates P < 0.01.

Figure 25, comprising Figures 25A and 25B, is two graphs demonstrating the anti-tumor activity of BCMA CART cells in the KMS-11 human multiple myeloma model in two independent experiments. The mean bioluminescence (+/- SEM) of tumor cells shows the disease burden in all animals, expressed as photons per second (p/s) (or total flux or BLI) for all mice. The significance of day 28 relative to the vehicle was calculated by ANOVA; * in Fig. 25A indicates P < 0.01. In Figure 25B, BCMA-4NP* represents the BCMA-4NP results from the first experiment (results shown in Figure 25A).

Figure 26, comprising Figures 26A, 26B, 26C and 26D, is a graph showing the proliferation of BCMA-CART cells by quantifying the number of BCMA-CART cells in peripheral blood of KMS-11-luc tumor-loaded mice. After treatment with CART cells, peripheral blood T cells were analyzed weekly on days 1, 3, 7, 10, and 14, and thereafter. From the first tumor experiment (the results shown in Figure 25A), the CD4+ CART population was evaluated in Figure 26A and the CD8+ CART population was evaluated in Figure 26B. From the second tumor experiment (the results shown in Figure 25B), the CD4+ CART population was evaluated in Figure 26C and the CD8+ CART population was evaluated in Figure 26D.

Figure 27, comprising Figures 27A, 27B, 27C and 27D, is a graph showing the expansion of T cells expressing BCMA CAR in bone marrow and spleen at the end of the first tumor experiment (results shown in Figure 25A). The average number of CD4+ T cells expressing BCMA CAR in bone marrow (Fig. 27A) and spleen (Fig. 27B) was calculated. Calculate CD4=8+ performance BCMA in bone marrow (Fig. 27C) and spleen (Fig. 27D) The average number of T cells in CAR. The J6MO sample represents CART cells expressing the BCMA-4NP CAR construct.

Figure 28, comprising Figures 28A and 28B, is a graph showing the lentiviral power valence of selected BCMA CAR constructs in two independent lentivirus experiments. In the first round of testing, two different DNA preparations (A and B) of the BCMA CAR construct were tested (Fig. 28A). In the second round of testing, three different DNA preparations (A, B and C) of the BCMA CAR construct were tested (Figure 28B).

Figure 29 is a graph showing the competition analysis between BCMA-4NP and selected BCMA CAR constructs BCMA-4 (B4), BCMA-10 (B10), BCMA-13 (B13), and BCMA-15 (B15).

Figure 30, comprising Figures 30A, 30B, 30C, 30D and 30E, is a graph showing the results of affinity analysis of selected BCMA constructs: BMCA-10 (Figure 30A); BCMA-13 (Figure 30B), BCMA-15 ( Figure 30C), BCMA-4 (Figure 30D) and BCMA-4NP (Figure 30E).

Figure 31 is a graph showing the selective binding of T cells of the selected BCMA CAR to recombinant BCMA. The recombinant form of BCMA and the closely related family members BAFFR and TACI comprising the protein fused to the Fc domain were incubated with T cells expressing BCMA-4, BCMA-10, BCMA-13 and BCMA-15. The percentage of cells bound to the recombinant protein (% of positive cells) was detected.

Figure 32, comprising Figures 32A, 32B, 32C, 32D, 32E and 32F, is a series of images depicting immunohistochemical staining of BCMA in brain tissue. BCMA staining in the creeping fibers of the cynomolgus macaque cerebellum (Fig. 32A). BCMA staining in neuronal cell bodies in the lower olive nucleus of cynomolgus monkeys (Fig. 32B). BCMA staining (Fig. 32C) and Ig staining (control) in the medulla of cynomolgus monkeys (Fig. 32E). BCMA staining (Fig. 32D) and Ig staining (control) in human medulla oblongata (Fig. 32F).

Figure 33, comprising Figures 33A, 33B, 33C, 33D and 33E, is a series of images and graphs depicting RNA analysis of BCMA expression in brain tissue. In situ hybridization of BCMA, DAPB and PPIB RNA in the cerebellum of non-human primates (Fig. 33A). Non-human primate medullary BCMA, DAPB and PPIB RNA in situ hybridization (Fig. 33B). Human cerebellum Quantitative PCR analysis of BCMA in the marrow, stomach and kidney (Fig. 33C). Quantitative PCR analysis of BCMA in white, gray, medulla, stomach and kidney of cynomolgus monkeys (Fig. 33D). RNAseq analysis of normal tissues in humans (Fig. 33E); boxes indicate BCMA expression in the cerebellum.

Figure 34 is a schematic diagram showing a timeline for evaluating the safety and feasibility of BCMA CART cell therapy in relapsed and/or refractory myeloma.

Figures 35A and 35B are graphs showing the concentration of cytokines secreted by BCMA-10 CART when co-cultured with target cells. Figure 35A is a graph showing the concentrations of interleukin-2 (IL-2) and interferon-gamma (IFNy) secreted by BCMA-10 CART. Figure 35B is a graph showing the concentration of tumor necrosis factor-α (TNF-α) secreted by BCMA-10 CART.

36A and 36B are graphs/graphs showing growth curves and efficiencies of huBCMA-BBz lentiviral transduction of T cells. Figure 36A is a graph showing the number of T cells transduced by the huBCMA-BBz vector several days after amplification. Figure 36B is a set of flow cytometry showing the performance of BCMA ex vivo expansion on day 6 of CART-BCMA cells (T cells transduced by huBCMA-BBz vector) compared to untransduced NTD cells. Graph.

Figure 37 is a graph showing flow cytometry histograms of BCMA surface expression on various cell lines, including K562-BCMA cells and multiple myeloma cell lines NCI H929, U266, RPMI 8226, OPM2, and MM1S. For all graphs, an orange solid peak indicates an isotype control and a blue solid peak was stained with a BCMA antibody.

Figures 38A and 38B are graphs showing the concentration of cytokines produced by CART-BCMA cells in response to myeloma cell lines. Figure 38A shows the concentration of IL-2 produced, and Figure 38B shows the concentration of IFN-[gamma produced. Values represent cytokine concentrations in pg/mL.

Figures 39A, 39B and 39C are graphs showing antigen-specific killing of BCMA ⁺ multiple myeloma cell lines by CART-BCMA cells. Figure 39A shows antigen-specific killing of K562-BCMA cells, Figure 39B shows antigen-specific killing of RPMI 8226 cells, and Figure 39C shows antigen-specific killing of MM1S cells.

Figures 40A, 40B and 40C are graphs showing that CART-BCMA cells show potent anti-myeloma activity in vivo. Figure 40A is a graph showing total radiation in untransduced mice, and Figure 40B is a graph showing total radiation in CART-BCMA mice. Figure 40C is a graph showing the percent survival of NTD or CART-BCMA mice after injection of T cells. The backlight emission from the RPMI 8226CBG+ tumor was shown, with individual animals depicted in gray and the median total radiation amount displayed in red. Each group has n=10. Time is shown by the number of weeks after T cell injection.

Figures 41A-41D show various configurations on a single vector, such as where the U6 regulated shRNA is upstream or downstream of the EF1 alpha regulated CAR coding element. In the exemplary constructs depicted in Figures 41A and 41B, transcription is carried out in the same direction via the U6 and EF1 alpha promoters. In the exemplary constructs depicted in Figures 41C and 41D, transcription is carried out in different directions via the U6 and EF1 alpha promoters. In Figure 41E, the shRNA (and the corresponding U6 promoter) are on the first vector and the CAR (and the corresponding EF1 alpha promoter) are on the second vector.

Figure 42 depicts the structure of two exemplary RCAR configurations. Antigen binding members comprise an antigen binding domain, a transmembrane domain, and a exchange domain. Intracellular binding members comprise a exchange domain, a co-stimulatory signaling domain, and a major signaling domain. Both configurations demonstrate that the first and second exchange domains described herein can be oriented differently with respect to antigen binding members and intracellular binding members. This document further describes other RCAR configurations.

Figure 43 shows that proliferation of transduced T cells expressing CAR in a cell culture system is enhanced by low dose RAD001. CART was co-cultured with Nalm-6 cells in the presence of different concentrations of RAD001. The number of CAR-positive CD3-positive T cells (black) and total T cells (grey) was assessed on day 4 after co-culture.

Figure 44 depicts tumor growth measurements of RAD001 or NAMV6-luc cells administered daily at 0.3, 1, 3, and 10 mg/kg (mpk). Circles refer to mediators; squares refer to 10 mg/kg doses of RAD001; triangles refer to 3 mg/kg doses of RAD001, inverted triangles refer to 1 mg/kg doses of RAD001; and diamonds refer to 0.3 mg/kg doses of RAD001.

Figures 45A and 45B show pharmacokinetic profiles showing the amount of RAD001 in the blood of NSG mice with NALM6 tumors. Figure 45A shows the PK on day 0 after the first RAD001 is given. Figure 45B shows the 14th day PK after the final RAD001 is given. The diamond refers to the 10 mg/kg dose of RAD001; the square refers to the 1 mg/kg dose of RAD001; the triangle refers to the 3 mg/kg dose of RAD001; and the x refers to the 10 mg/kg dose of RAD001.

Figures 46A and 46B show in vivo proliferation of humanized CD19 CART cells administered with and without RAD001. Daily low doses of RAD001 (0.003 mg/kg) caused an increase in CAR T cell proliferation, exceeding the normal level of huCAR19 proliferation. Figure 46A shows CD4 ⁺ CAR T cells; Figure 46B shows CD8 ⁺ CAR T cells. Circles refer to PBS; squares refer to huCTL019; triangles refer to huCTL019 using 3 mg/kg RAD001; inverted triangles refer to huCTL019 using 0.3 mg/kg RAD001; diamonds refer to huCTL019 using 0.03 mg/kg RAD001; and circles refer to Use huCTL019 of 0.003 mg/kg RAD001.

Figure 47 depicts CD19 expression in patient tumor cells. CD138 ⁺ CD45 ^dark tumor cells were stained for CD19 (x-axis) and CD38 (y-axis). About 1-2% of tumor cells express CD19 antigen.

definition

All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, unless otherwise defined.

The term "a" and "an" refers to a grammatical object of one or more (ie, at least one) items. By way of example, "an element" means an element or more than one element.

When referring to measurable values such as quantity, duration, and the like, the term "about" is intended to cover ± 20% compared to the specified value, or in some cases ± 10%, or in some cases ± 5%. Or, in some cases ±1%, or in some cases ±0.1%, and thus the variation is suitable for performing the disclosed method.

The term "chimeric antigen receptor" or "CAR" refers to a cytoplasmic signaling structure comprising at least an extracellular antigen binding domain, a transmembrane domain, and a functional signaling domain comprising a stimulatory molecule as defined below. Recombinant polypeptide constructs of domains (also referred to herein as "intracellular signaling domains"). In some embodiments, the domain in the CAR polypeptide construct is in the same polypeptide chain, for example, comprising a chimeric fusion protein. In some embodiments, the domains in the CAR polypeptide construct are not adjacent to each other, such as in a different polypeptide chain, such as provided in RCAR as described herein.

In one aspect, the stimulatory molecule of CAR is an ξ chain associated with a T cell receptor complex. In one aspect, the cytoplasmic signaling domain comprises a major signaling domain (eg, the major signaling domain of CD3-ξ). In one aspect, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule as defined below. In one aspect, the costimulatory molecule is selected from the group consisting of 4-1BB (i.e., CD137), CD27, ICOS, and/or CD28. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and a functional signaling domain derived from a costimulatory molecule and a functional derived from a stimulatory molecule The intracellular signaling domain of the signaling domain. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and a functional signaling domain comprising two one or more costimulatory molecules and a An intracellular signaling domain derived from a functional signaling domain of a stimulating molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and a functional signaling domain comprising at least two derived from one or more costimulatory molecules and An intracellular signaling domain derived from a functional signaling domain of a stimulatory molecule. In one aspect, CAR is at the amine end of the CAR fusion protein (N End) contains a preamble sequence as appropriate. In one aspect, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen recognition domain, wherein the leader sequence is cleaved from the antigen recognition domain (eg, aa scFv) as appropriate during cell processing and CAR localization to the cell membrane.

A CAR comprising an antigen binding domain (eg, an scFv, a single domain antibody or a TCR (eg, a TCR alpha binding domain or a TCR beta binding domain)) that targets a particular tumor marker X is also referred to as XCAR, wherein X can be as described herein Tumor marker. For example, a CAR comprising an antigen binding domain that targets BCMA is referred to as BCMA CAR. CAR can be expressed in any cell, such as an immune effector cell (e.g., a T cell or an NK cell) as described herein.

The term "signaling domain" refers to a protein function that functions by transmitting information within a cell to regulate cellular activity via a defined signaling pathway by generating a second messenger or by acting as an effector in response to such messenger. section.

As used herein, the term "BCMA" refers to a B cell maturation antigen. BCMA (also known as TNFRSF17, BCM or CD269) is a member of the tumor necrosis receptor (TNFR) family and is predominantly expressed on terminally differentiated B cells, such as memory B cells and plasma cells. Its ligand is called the B cell activating factor (BAFF) and the proliferative inducing ligand (APRIL) of the TNF family. BCMA is involved in mediating the survival of plasma cells to maintain long-term humoral immunity. The BCMA gene is encoded on chromosome 16 to produce a 994 nucleotide long primary mRNA transcript (NCBI-registered NM_001192.2) encoding 184 amino acid proteins (NP_001183.2). A second antisense transcript derived from the BCMA locus has been described which can play a role in the regulation of BCMA expression (Laabi Y. et al, Nucleic Acids Res., 1994, 22: 1147-1154). Additional transcript variants have been described with unknown significance (Smirnova AS et al. Mol Immunol., 2008, 45(4): 1179-1183). A second isoform has been identified, also known as TV4 (Uniprot identifier Q02223-2). As used herein, "BCMA" includes proteins comprising full length wild-type BCMA mutations, such as point mutations, fragments, insertions, deletions, and splice variants.

The term "antibody" as used herein refers to an immunogen derived from a specific binding antigen. A protein or polypeptide sequence of a globulin molecule. The antibody may be multi- or single-strand, multi-stranded or single-stranded or intact immunoglobulin and may be derived from natural or recombinant sources. The antibody can be a tetramer of immunoglobulin molecules.

The term "antibody fragment" refers to at least a portion of an intact antibody or a recombinant variant thereof and refers to an antigen binding domain sufficient for the antibody fragment to recognize and specifically bind to a target such as an antigen, eg, an antigenic determining variable region of an intact antibody. . Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab') ₂ and Fv fragments, scFv antibody fragments, linear antibodies, single domain antibodies such as sdAbs (VL or VH), Camelidae VHH domains and By other antigenic determinant binding fragments, such as comprising two or more, for example two Fab fragments joined by a disulfide bridge in the hinge region, or two or more, for example two isolated CDRs or linked antibodies A multispecific antibody formed by an antibody fragment of a bivalent fragment. Antibody fragments can also be incorporated into single domain antibodies, maximal antibodies, minibodies, nanobodies, intrabodies, bifunctional, trifunctional, tetrafunctional, v-NAR, and double scFv (see, for example, Hollinger and Hudson, Nature Biotechnology 23: 1126-1136, 2005). Antibody fragments can also be grafted to a polypeptide-based backbone, such as a type III fibronectin (Fn3) (see U.S. Patent No. 6,703,199, which describes a fibronectin polypeptide minibody).

The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, wherein the light and heavy chain variable regions are via a short flexible polypeptide The linker is contiguously linked and can behave as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, an scFv can have a VL and VH variable region in either order, eg, relative to the N-terminus and C-terminus of the polypeptide, the scFv can comprise a VL-linker-VH or can comprise a VH-linkage Sub-VL.

The term "complementarity determining region" or "CDR" as used herein refers to an amino acid sequence that confers antigen specificity and binding affinity in the variable region of an antibody. For example, typically, three CDRs (eg, HCDR1, HCDR2, and HCDR3) are present in each heavy chain variable region and three CDRs (LCDR1, LCDR2, and LCDR3) in each of the light chain variable regions. Established CDR The precise amino acid sequence boundaries can be used in any of a number of well known protocols, including Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th Edition Public Health Service, National Institutes of Health, Bethesda, MD ( The "Kabat" numbering scheme, Al-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme) described in the scheme or a combination thereof. According to the Kabat numbering scheme, in some embodiments, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3). And the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). According to the Chothia numbering scheme, in some embodiments, the CDR amino acids in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in VL The base numbers are 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). In a combined Kabat and Chothia numbering scheme, in some embodiments, the CDRs correspond to amino acid residues that are part of the Kabat CDR, the Chothia CDR, or both. For example, in some embodiments, the CDRs correspond to VH, such as a mammalian VH, such as amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH. And VL, such as mammalian VL, such as amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL.

Portions of the CAR compositions of the invention comprising antibodies or antibody fragments thereof can exist in a variety of forms, for example, wherein the antigen binding domain acts as a polypeptide chain (including, for example, a single domain antibody fragment (sdAb), a single chain antibody (scFv), or, for example, humanization. Partial performance of antibodies) (Harlow et al, 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al, 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al, 1988, Proc. Natl. Acad. Sci. USA 85: 5879-5883; Bird et al, 1988, Science 242: 423-426). In one aspect, the antigen binding domain of the CAR composition of the invention comprises an antibody Fragment. In another aspect, the CAR comprises an antibody fragment comprising an scFv.

As used herein, the term "binding domain" or "antibody molecule" (also referred to herein as "anti-target (eg, BCMA) binding domain") refers to a protein comprising at least one immunoglobulin variable domain sequence. For example, an immunoglobulin chain or a fragment thereof. The term "binding domain" or "antibody molecule" encompasses antibodies and antibody fragments. In one embodiment, the antibody molecule is a multispecific antibody molecule, eg, comprising a plurality of immunoglobulin variable domain sequences, wherein the first of the plurality of immunoglobulin variable domain sequences has a first antigen The binding specificity of the base is determined and the second immunoglobulin variable domain sequence of the plurality has binding specificity for the second epitope. In one embodiment, the multispecific antibody molecule is a bispecific antibody molecule. Bispecific antibodies are specific for no more than two antigens. The bispecific antibody molecule is characterized in that the first immunoglobulin variable domain sequence has binding specificity for the first epitope and the second immunoglobulin variable domain sequence has binding specificity for the second epitope Sex. The term "antibody heavy chain" refers to the larger of the two types of polypeptide chains that are present in the antibody molecule in a naturally occurring configuration and that typically determine the class to which the antibody belongs.

The term "antibody light chain" refers to the lesser of the two types of polypeptide chains present in the antibody molecule in a naturally occurring configuration. The Kappa (κ) and Lambda (λ) light chains refer to the two major antibody light chain isotypes.

The term "recombinant antibody" refers to an antibody produced using recombinant DNA techniques, such as antibodies expressed by phage or yeast expression systems. The term is also understood to mean an antibody produced by synthesizing a DNA molecule encoding an antibody (and the DNA molecule exhibits an antibody protein) or an amino acid sequence of a specified antibody, wherein the DNA or amino acid sequence has been used. Recombinant DNA or amino acid sequence techniques available and well known in the art are available.

The term "antigen" or "Ag" refers to a molecule that causes an immune response. This immune response can involve antibody production or specific immunity to the activation of the cells or both. Those skilled in the art will appreciate that any macromolecule comprising almost all proteins or peptides can serve as an antigen. this In addition, the antigen can be derived from recombinant or genomic DNA. It will be understood by those skilled in the art that when the term is used herein, any DNA comprising a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an immune response thus encodes an "antigen." Moreover, those skilled in the art will appreciate that the antigen need not be encoded solely by the full length nucleotide sequence of the gene. It will be apparent that the invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and such nucleotide sequences are arranged in various combinations to encode a polypeptide that elicits a desired immune response. In addition, those skilled in the art should understand that antigens do not have to be encoded by "genes" at all. It will be apparent that the antigen may be synthetically produced or may be derived from a biological sample or may be a macromolecule other than a polypeptide. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells, or body fluids with other biological components.

The term "anti-tumor effect" refers to an biological effect that can be visualized by various means including, but not limited to, for example, a reduction in tumor volume, a decrease in the number of tumor cells, a decrease in the number of cancer metastases, an increase in life expectancy, and a decrease in tumor cell proliferation. The survival rate of tumor cells is reduced, or various physiological symptoms associated with cancerous conditions are improved. The "anti-tumor effect" can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies of the present invention to prevent the first appearance of a tumor.

The term "anticancer effect" refers to an biological effect that can be manifested by various means including, but not limited to, for example, a reduction in tumor volume, a decrease in the number of tumor cells, a decrease in the number of cancer metastases, an increase in life expectancy, and a decrease in cancer cell proliferation. The survival rate of cancer cells is reduced, or various physiological symptoms associated with cancerous conditions are improved. "Anti-cancer effects" can also be manifested by the ability of peptides, polynucleotides, cells and antibodies to prevent the first appearance of cancer. The term "anti-tumor effect" refers to an biological effect that can be manifested by a variety of means including, but not limited to, for example, a reduction in tumor volume, a decrease in the number of tumor cells, a decrease in tumor cell proliferation, or a decrease in tumor cell survival. The term "autologous" refers to any material derived from the same individual as the individual that can subsequently be reintroduced.

The term "allogene" refers to any material derived from a different animal of the same species as the individual into which the material will be introduced. When the genes of one or more loci are different, two or two More than one individual is referred to as an allogeneic species. In some aspects, allogeneic materials from individuals of the same species may be genetically distinct to interact in an antigenic manner.

The term "heterologous" refers to an animal from which the graft is derived from a different species.

The term "scavenging" as used herein refers to an art-recognized in vitro process by which a donor or patient's blood is removed from a donor or patient and by separating the selected specific components from the device and, for example. The remaining material is returned to the circulation of the donor or patient by retransfusion. Therefore, in the case of "scavenging a sample", it refers to a sample obtained by using a scavenging technique.

The term "combination" means a fixed combination in a unit dosage form, or wherein the compound of the invention and the combination partner (eg, another drug as described below, also referred to as "therapeutic agent" or "adjuvant") can be simultaneously independent. The administration is administered separately or separately during the time interval, especially where such time intervals allow the combination of collocations to exhibit collaboration, such as a combination of synergistic effects. A single component can be packaged in a kit or packaged separately. One or both of the pre-administration components (eg, powder or liquid) can be reconstituted or diluted to the desired dose. As used herein, the terms "co-administered" or "combined administration" or the like are intended to encompass the application of a selected combination to a single individual (eg, a patient) in need thereof, and are intended to include that the agent is not necessarily Treatment regimens with the same route of administration or concurrent administration. As used herein, the term "pharmaceutical combination" means both a product produced by mixing or combining more than one active ingredient and includes both fixed and unfixed combinations of the active ingredients. The term "fixed combination" means that the active ingredient, for example, a compound of the invention and a combination compositing, are administered to a patient simultaneously in a single entity or dosage form. The term "non-fixed combination" means that the active ingredient, for example, a compound of the present invention and a combination partner, are administered to a patient simultaneously, in parallel or sequentially in separate physical forms, without specific time constraints, wherein such administration is in a patient. Two compounds that provide therapeutically effective levels. The latter also applies to combination therapies, for example the administration of three or more active ingredients.

The term "cancer" refers to a disease characterized by rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or spread to other parts of the body via the bloodstream and lymphatic system. Examples of various cancers are described herein and include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, Lung cancer and its similar cancer. Preferred cancers to be treated by the methods described herein include multiple myeloma, Hodgkin's lymphoma or non-Hodgkin's lymphoma.

The terms "tumor" and "cancer" are used interchangeably herein, for example, two terms encompass both entities and fluids, such as diffuse or circulating tumors. As used herein, the term "cancer" or "tumor" includes pre-malignant and malignant cancers and tumors.

The term indicates the relationship between the first and second molecules when "derived from" is used herein. It generally refers to the structural similarity between the first molecule and the second molecule and does not encompass or include a process or source limitation on the first molecule derived from the second molecule. For example, in the case of an intracellular signaling domain derived from a CD3 ζ molecule, the intracellular signaling domain retains sufficient CD3 ζ structure to have the desired function, i.e., the ability to generate a signal under appropriate conditions. It does not cover or include limitations on the specific process of generating an intracellular signaling domain, for example, it does not mean that in order to provide an intracellular signaling domain, it is necessary to start with a CD3ζ sequence and delete unnecessary sequences, or apply a mutation to achieve Intracellular signaling domain.

The phrase "disease associated with BCMA performance" includes, but is not limited to, diseases associated with cells expressing BCMA (eg, wild-type or mutant BCMA) or conditions associated with cells expressing BCMA (eg, wild-type or mutant BCMA). , including, for example, proliferative diseases such as cancer or malignant diseases or precancerous conditions, such as myelodysplasia, myelodysplastic syndromes or leukemia prodromes; or non-cancers associated with cells expressing BCMA (eg, wild-type or mutant BCMA) Related indications. For the avoidance of doubt, diseases associated with BCMA performance may include, for example, because BCMA has been down-regulated due to, for example, treatment with a molecule that targets BCMA (eg, a BCMA inhibitor described herein), but currently does not exhibit BCMA, but has previously exhibited BCMA Cell-related conditions. In one aspect, with BCMA (eg wild The type-related or mutant BCMA) expression-related cancer is blood cancer. In one aspect, the blood cancer is leukemia or lymphoma. In one aspect, a cancer associated with BCMA (eg, wild-type or mutant BCMA) expression is a malignant disease of differentiated plasma B cells. In one aspect, cancers associated with BCMA (eg, wild-type or mutant BCMA) include cancer and malignant diseases including, but not limited to, for example, one or more acute leukemias including, but not limited to, for example, B-cell acute lymphoid Leukemia ("BALL"), T-cell acute lymphoblastic leukemia ("TALL"), acute lymphocytic leukemia (ALL); one or more chronic leukemias, including but not limited to, for example, chronic myelogenous leukemia (CML), chronic Lymphocytic leukemia (CLL). Other cancer or hematological conditions associated with the performance of BMCA (eg, wild-type or mutant BCMA) include, but are not limited to, for example, B-cell pro-lymphocytic leukemia, parental plasmacytoid dendritic cell tumor, primary Lycoma lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative condition, MALT lymphoma, mantle cell lymphoma, Marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell tumor, Waldenstrom's giant ball Proteinemia and "leukemia prodrosis", leukemia prodrosis is a variety of blood conditions caused by the ineffective (or dysplasia) of bone marrow blood cells; and the like. In some embodiments, the cancer is multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, or glioblastoma. In the examples, diseases associated with BCMA manifestations include plasma cell proliferative disorders such as asymptomatic myeloma (and suspicion of multiple myeloma or inert myeloma), unexplained gamma globulinemia (MGUS), var. Denver's macroglobulinemia, plasmacytoma (eg plasma cell cachexia, solitary myeloma, solitary plasmacytoma, extramedullary plasmacytoma and multiple plasmacytoma), systemic amyloid light chain Amyloidosis and POEMS syndrome (also known as Crohn-Fox syndrome, sorghum and PEP syndrome). Other diseases associated with BCMA (eg, wild-type or mutant BCMA) include, but are not limited to, atypical and/or, for example, related to BCMA (eg, wild-type or mutant BCMA) performance. Non-classical cancer, malignant disease, precancerous condition or proliferative disease, such as a cancer as described herein, such as prostate cancer (eg, castration resistant or therapeutic resistant prostate cancer, or metastatic prostate cancer), pancreatic cancer, or lung cancer .

And the BCMA (e.g. wild-type or mutant BCMA) related to the non-cancer-related conditions include viral infections; such as HIV, fungal infections (e.g. Cryptococcus neoformans (C.neoformans)); autoimmune diseases, such as rheumatoid arthritis, Systemic lupus erythematosus (SLE or lupus), pemphigus vulgaris and Sjogren's syndrome; inflammatory bowel disease, ulcerative colitis; transplant-associated allo-specific immune disease associated with mucosal immunity; Among them, humoral immunity is an important immune response to biological agents (such as factor VIII). In embodiments, non-cancer related indications associated with BCMA performance include, but are not limited to, for example, autoimmune diseases (eg, lupus), inflammatory conditions (allergy and asthma), and transplantation. In some embodiments, the cell expressing the tumor antigen exhibits or at any time represents an mRNA encoding a tumor antigen. In one embodiment, the cell expressing the tumor antigen produces a tumor antigen protein (eg, a wild type or mutation) and the tumor antigen protein can be present at a normal or reduced level. In one embodiment, the cells expressing the tumor antigen produce a detectable level of tumor antigen protein at a time point and subsequently produce a substantially undetectable tumor antigen protein.

The term "conservative sequence modification" means that the amino acid modification does not significantly affect or alter the binding characteristics of the antibody or antibody fragment containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies or antibody fragments of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A conservative substitution is the substitution of an amino acid residue with an amino acid residue having a similar side chain. A family of amino acid residues with similar side chains have been defined in the art. Such families include basic side chains (eg, lysine, arginine, histidine), acidic side chains (eg, aspartic acid, glutamic acid), and no polar side chains (eg, glycine) , aspartame, glutamic acid, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (eg Such as alanine, valine, leucine, isoleucine, valine, phenylalanine, methionine, β-branched side chains (such as sulphate, valine, isoleucine) and Amino acid of an aromatic side chain (eg, tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the CAR of the invention can be substituted with other amino acid residues of the same side chain family and the altered CAR can be tested using the functional assays described herein.

The term "stimulating" refers to the binding of a stimulatory molecule (eg, a TCR/CD3 complex) to its cognate ligand, thereby mediating a signal transduction event (such as, but not limited to, signal transduction via a TCR/CD3 complex). The main reaction induced. Stimulation can mediate changes in the performance of certain molecules, such as down-regulation of TGF-[beta], and/or reorganization of cytoskeletal structures and the like.

The term "stimulating molecule" refers to a molecule expressed by a T cell that provides a major cytoplasmic signaling sequence that modulates the primary activation of the TCR complex in a stimulatory manner for at least some aspects of the T cell signaling pathway. In some embodiments, the IT-containing MIAM domain reproduces signaling independent of the major TCR of the endogenous TCR complex. In one aspect, primary signaling is initiated by binding, for example, a TCR/CD3 complex to a peptide-loaded MHC molecule, and which causes mediated T cell responses including, but not limited to, proliferation, activation, differentiation, and Its analogues. The major cytoplasmic signaling sequence (also referred to as the "primary signaling domain") that acts in a stimulatory manner may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM. Examples of ITAM-containing major cytoplasmic signaling sequences specifically for use in the present invention include, but are not limited to, those derived from: TCR ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS"), FcεRI and CD66d, DAP10 and DAP12. In one of the specific CARs of the invention, the intracellular signaling domain of any one or more of the CARs of the invention comprises an intracellular signaling sequence, such as the major signaling sequence of CD3-ζ. In one of the specific CARs of the invention, the major signaling sequence of CD3-ζ is the sequence provided in the form of SEQ ID NO: 9, or from a non-human species (eg, mouse, rodent, monkey, donkey, and the like). The equivalent residue of ). In one of the specific CARs of the present invention, the main signal of CD3-ζ The conduction sequence is the sequence as provided in SEQ ID NO: 10, or an equivalent residue from a non-human species (eg, mouse, rodent, monkey, sputum, and the like). The term "antigen-presenting cell" or "APC" refers to an immune system cell that exhibits a foreign antigen complexed with a major histocompatibility complex (MHC) on its surface, such as helper cells (eg, B cells, dendritic cells, and the like). cell). T cells can recognize these complexes using their T cell receptor (TCR). APC treats the antigen and presents it to T cells.

The term "intracellular signaling domain" as used herein refers to the intracellular portion of a molecule. In an embodiment, the intracellular signaling domain transduces an effector function signal and directs the cell to perform a specialized function. Although the entire intracellular signaling domain is typically employed, in most cases it is not necessary to use the entire chain. To the extent that truncated portions of the intracellular signaling domain are used, such truncated portions can be used to replace the entire strand as long as the effector function signal is transduced. Thus the term intracellular signaling domain is meant to include any truncated portion of the intracellular signaling domain sufficient to transduce an effector function signal.

The intracellular signaling domain produces a signal that promotes the immune effector function of cells containing CAR, such as CART cells. Examples of immune effector functions (e.g., in CART cells) include cytolytic activity and helper activity, including secretion of cytokines.

In an embodiment, the intracellular signaling domain can comprise a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from molecules responsible for major stimulation or antigen dependent modeling. In an embodiment, the intracellular signaling domain can comprise a co-stimulatory intracellular domain. Exemplary co-stimulatory intracellular signaling domains include those derived from molecules responsible for costimulatory signals or antigen-independent stimuli. For example, in the case of CART, the major intracellular signaling domain may comprise a cytoplasmic sequence of a T cell receptor, and the co-stimulated intracellular signaling domain may comprise a cytoplasmic sequence from a helper or co-stimulatory molecule .

The major intracellular signaling domain may comprise a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM. Major cytoplasmic signaling sequence containing ITAM Examples include, but are not limited to, those derived from: CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (also known as “ICOS”), FcεRI, CD66d, DAP10, and DAP12 .

The terms "ζ" or "ζ", "CD3-ζ" or "TCR-ζ" are defined as proteins supplied as GenBan Deposit No. BAG36664.1 or from non-human species (eg mice, rodents, monkeys, baboons and The equivalent residue of a similar species, and the "ζ stimulating domain" or "CD3-ζ stimulating domain" or "TCR-ζ stimulating domain" is defined as the amino acid residue from the cytoplasmic domain of the ζ chain, It is sufficient to functionally transmit the initial signal necessary for T cell activation. In one aspect, the cytoplasmic domain of sputum contains residues 52 to 164 of Genbank accession number BAG36664.1 or non-human species (eg, mouse, rodent, monkey, pupa and its functional orthologs) Equivalent residue of a similar species). In one aspect, the "ζ stimulation domain" or "CD3-ζ stimulation domain" is the sequence provided in SEQ ID NO: 9. In one aspect, the "ζ stimulation domain" or "CD3-ζ stimulation domain" is the sequence provided in SEQ ID NO: 10.

The term "co-stimulatory molecule" refers to a homologous binding partner on a T cell that specifically binds to a co-stimulatory ligand whereby T cells mediate a co-stimulatory response such as, but not limited to, proliferation. A costimulatory molecule is a cell surface molecule other than an antigen receptor or a ligand thereof that is required for an effective immune response. Costimulatory molecules include, but are not limited to, MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activating molecules (SLAM proteins), activated NK cell receptors, BTLA, Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3, CDS , ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 ( NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a and specific binding to CD83 Body.

Co-stimulation of the intracellular signaling domain refers to the intracellular portion of a costimulatory molecule.

The intracellular signaling domain may comprise all of the intracellular portion or all of the native intracellular signaling domain of the molecule from which it is derived, or a functional fragment thereof.

The term "4-1BB" refers to a member of the TNFR superfamily that is provided by the amino acid sequence in GenBank Accession Number AAA62478.2, or from non-human species (eg, mice, rodents, monkeys, baboons, and the like). Residue; and the "4-1BB co-stimulatory domain" is defined as the amino acid residue 214-255 of GenBank Accession Number AAA62478.2, or from non-human species (eg, mouse, rodent, monkey, pupa and its Equivalent residue of a similar species). In one aspect, the "4-1BB costimulatory domain" is the sequence provided by SEQ ID NO: 7 or an equivalent residue from a non-human species (eg, mouse, rodent, monkey, ape, and the like) base.

"Immune effector cell" as used herein, refers to a cell that is involved in an immune response, such as an immune response. Examples of immune effector cells include T cells such as α/β T cells and γ/δ T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and bone marrow-derived phagocytic cells.

"Immune effector function or immune effector" as used herein, refers to, for example, the function of an immune effector cell to enhance or promote the immune attack of a target cell or should. For example, an immune effector function or response refers to a property in which T or NK cells promote killing or growth or proliferation inhibition of a target cell. In the case of T cells, primary stimulation and co-stimulation are examples of immune effector functions or responses.

The term "effector function" refers to the specialized function of a cell. The effector function of T cells can be, for example, cytolytic activity or helper activity, including secretion of cytokines.

The term "coding" refers to the intrinsic property of a particular sequence of nucleotides in a polynucleotide such as a gene, cDNA or mRNA that acts as a template for the synthesis of other polymers and macromolecules in biological processes, such polymers and macromolecules. A defined nucleotide sequence (eg, rRNA, tRNA, and mRNA) or a defined amino acid sequence and biological properties obtained thereby. Thus, if the transcription and translation of mRNA corresponding to a gene produces a protein in a cell or other biological system, the gene, cDNA or RNA encodes the protein. A non-coding strand of a nucleotide sequence that is identical to the mRNA sequence and is typically provided in the sequence listing and used as a template for transcription of the gene or cDNA may be referred to as a protein or gene or cDNA.

Unless otherwise specified, "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences which are degenerate forms of each other and which encode the same amino acid sequence. To the extent that a nucleotide sequence encoding a protein may contain an intron in some versions, the term nucleotide sequence encoding a protein or RNA may also include an intron.

The terms "effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to an amount of a compound, formulation, substance, or composition that is effective to achieve a particular biological result as described herein.

The term "endogenous" refers to any substance produced from or within an organism, cell, tissue or system.

The term "exogenous" refers to any substance introduced or produced from outside a living organism, cell, tissue or system.

The term "expression" refers to the transcription and/or transcription of a particular nucleotide sequence driven by a promoter. Translation.

The term "transfer vector" refers to a composition of matter comprising an isolated nucleic acid and which can be used to deliver an isolated nucleic acid to the interior of a cell. Many vectors are known in the art and include, but are not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plastids, and viruses. Thus, the term "transfer vector" includes autonomously replicating plastids or viruses. The term should also be interpreted to further include aprotic and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, and the like. Examples of viral transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, and the like.

The term "expression carrier" refers to a vector comprising a recombinant polynucleotide comprising a expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all known in the art, including viscososomes, plastids (eg, naked or contained in liposomes), and viruses (eg, lentiviruses, retroviruses, adenoviruses, and Adeno-associated virus).

The term "llow virus" refers to a family of retroviral families. Lentiviruses are unique in retroviruses because they are capable of infecting undifferentiated cells; they can deliver a sufficient amount of genetic information to the DNA of the host cell, making it one of the most efficient methods of gene delivery vectors. HIV, SIV and FIV are all examples of lentiviruses.

The term "lentiviral vector" refers to a vector derived from at least a portion of a lentiviral genome, and particularly includes a self-inactivated lentivirus as provided by Milone et al., Mol. Ther. 17(8): 1453-1464 (2009). Carrier. Other examples of lentiviral vectors that can be used in the clinic include, but are not limited to, ENTIVECTOR® gene delivery technology from Oxford BioMedica, LENTIMAX ^(TM) vector systems from Lentigen, and the like. Non-clinical types of lentiviral vectors are also available and will be known to those skilled in the art.

The term "homologous" or "identity" refers to between two polymer molecules, for example Subunit sequence identity between two nucleic acid molecules such as two DNA molecules or two RNA molecules, or between two polypeptide molecules. When the position of a subunit of both molecules is occupied by the same monomer subunit; for example, if the position in each of the two DNA molecules is occupied by adenine, then it is homologous or identical at that position. . The homology between two sequences is a direct function of the number of matches or homologous positions; for example, if half of the positions in the two sequences (eg, five positions in a polymer of ten subunits) are homologous, then The two sequences are 50% homologous; if 90% of the positions (eg, 9 out of 10) match or are homologous, the two sequences are 90% homologous.

A "humanized" form of a non-human (eg, murine) antibody is a chimeric immunoglobulin, immunoglobulin chain or fragment thereof (such as Fv, Fab, Fab', F) containing minimal sequence derived from a non-human immunoglobulin. (ab') 2 or other antigen-binding sequence of the antibody). In most cases, humanized antibodies and antibody fragments thereof are human immunoglobulins (receptor antibodies or antibody fragments) in which residues from the complementarity determining regions (CDRs) of the recipient are replaced, for example, from having the desired specificity, Residues of CDRs of non-human species (donor antibodies) of mouse, rat or rabbit of affinity and ability. In some cases, the Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibody/antibody fragments can comprise residues that are neither present in the recipient antibody nor in the CDR or framework sequences of the input. Such modifications may further improve and optimize the efficacy of the antibody or antibody fragment. In general, a humanized antibody or antibody fragment thereof will comprise substantially all of at least one and typically two variable domains, wherein all or substantially all of the CDR regions correspond to and correspond to all regions of the non-human immunoglobulin Or most of the FR regions are those regions of the human immunoglobulin sequence. The humanized antibody or antibody fragment may also comprise at least a portion of an immunoglobulin constant region (Fc), which is typically the constant region of a human immunoglobulin. For further details, see Jones et al, Nature, 321 :522-525, 1986; Reichmann et al, Nature, 332:323-329, 1988; Presta, Curr. Op. Struct. Biol., 2: 593-596, 1992.

"Intact human" refers to an immunoglobulin, such as an antibody or antibody fragment, in which the entire molecule is of human origin or consists of an amino acid sequence consistent with the human form of the antibody or immunoglobulin.

The term "isolated" means changing or removing from a natural state. For example, a nucleic acid or peptide naturally present in a living animal is not "isolated," and the same nucleic acid or peptide that is partially or completely separated from a coexisting material in its native state is "isolated." The isolated nucleic acid or protein may be present in substantially purified form or may be present in a non-native environment such as a host cell.

In the context of the present invention, the following abbreviations of the commonly occurring nucleic acid bases are used. "A" means adenosine, "C" means cytosine, "G" means guanosine, "T" means thymidine and "U" means uridine.

The term "operably linked" or "transcriptional control" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence that results in the expression of a heterologous nucleic acid sequence. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. The operably linked DNA sequences can be contiguous with each other and in the same reading frame if necessary to join the two protein coding regions.

The term "parenteral" administration of an immunogenic composition includes, for example, subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.) or intrasternal injection, intratumoral or infusion techniques.

The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single or double-stranded form. Unless expressly limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (eg, degenerate codon substitutions, eg, conservative substitutions), dual genes, orthologs, SNPs, and Complement sequences and sequences that are clearly indicated. In particular, degenerate codon substitutions, such as conservative substitutions, may be substituted by a mixed base and/or a deoxyinosine residue by generating a third position in which one or more (or all) of the selected codons are selected. Sequence implementation (Batzer et al, Nucleic Acid Res. 19: 5081 (1991); Ohtsuka et al, J. Biol. Chem. 260: 2605-2608 (1985); and Rossolini et al, Mol. Cell. Probes 8: 91 -98 (1994)).

The terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a compound comprising an amino acid residue covalently linked by a peptide bond. The protein or peptide must contain at least two amino acids and is not limited to the maximum number of amino acids that can constitute a protein or peptide sequence. A polypeptide includes any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to short chains (which are also commonly referred to in the art as, for example, peptides, oligopeptides, and oligomers) and long chains (which are generally referred to in the art as proteins, which exist in a variety of types). ). "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins. Polypeptides include natural peptides, recombinant peptides, or a combination thereof.

The term "promoter" refers to a DNA sequence required for the specific transcription of a starting polynucleotide sequence, as recognized by the mechanism of cellular synthesis or the mechanism of introduction.

The term "promoter/regulatory sequence" refers to a nucleic acid sequence necessary for the expression of a gene product operably linked to a promoter/regulatory sequence. In some cases, the sequence can be a core promoter sequence and in other cases, the sequence can also include a booster sequence and other regulatory elements necessary for expression of the gene product. The promoter/regulatory sequence can be, for example, one that expresses the gene product in a tissue-specific manner.

The term "constitutive" promoter refers to a nucleotide sequence that, when operably linked to a nucleotide sequence encoding or indicating a gene product, causes a gene product to be produced in a cell under most or all of the physiological conditions of the cell.

The term "inducible" promoter means that when operably linked to a polynucleotide encoding or indicating a gene product, it is caused only when an inducer corresponding to the promoter is present in the cell. A nucleotide sequence that is substantially produced by a gene product in a cell.

The term "tissue-specific" promoter means that when operably linked to a polynucleotide encoding or indicating a gene, the gene product is caused substantially in the cell only when the cell is a cell of the tissue type corresponding to the promoter. The nucleotide sequence produced.

The term "cancer-associated antigen" or "tumor antigen" is used interchangeably to mean a molecule (usually a protein, carbohydrate or lipid) that is expressed on the surface of cancer cells, either in whole or in fragments (eg, MHC/peptide), and which is suitable for use in Pharmacological reagents preferentially target cancer cells. In some embodiments, the tumor antigen is a marker of normal cell and cancer cell expression, such as a lineage marker, such as CD19 on B cells. In some embodiments, the tumor antigen is a cell surface molecule that is overexpressed in cancer cells as compared to normal cells, such as 1x overexpression, 2x overexpression, 3x or more fold over normal cells which performed. In some embodiments, the tumor antigen is a cell surface molecule that is improperly synthesized in cancer cells, such as a molecule that contains a deletion, addition, or mutation compared to a molecule expressed on a normal cell. In some embodiments, the tumor antigen will be exclusively expressed on the cell surface of cancer cells, either in whole or in fragment form (eg, MHC/peptide), and will not be synthesized or expressed on the surface of normal cells. In some embodiments, a CAR of the invention comprises a CAR comprising an antigen binding domain (eg, an antibody or antibody fragment) that binds to an MHC-presenting peptide. Typically, peptides derived from endogenous proteins fill pockets of major histocompatibility complex (MHC) class I molecules and are recognized by T cell receptors (TCRs) on CD8+ T lymphocytes. The MHC class I complex is composed of all nucleated cells. In cancer, a virus-specific and/or tumor-specific peptide/MHC complex represents a unique class of cell surface targets for immunotherapy. TCR-like antibodies that target peptides derived from viral or tumor antigens in the context of human leukocyte antigen (HLA)-A1 or HLA-A2 have been described (see, for example, Sastry et al, J Virol. 2011 85(5): 1935- 1942; Sergeeva et al, Blood, 2011 117(16): 4262-4272; Verma et al, J Immunol 2010 184(4): 2156-2165; Willemsen et al, Gene Ther 2001 8(21): 1601-1608; Dao et al., Sci Transl Med 2013 5(176): 176ra33; Tassev et al., Cancer Gene Ther 2012 19(2): 84-100). For example, TCR-like antibodies can be identified by screening libraries, such as human scFv phage display libraries.

The term "tumor-supporting antigen" or "cancer-supporting antigen" is used interchangeably to refer to a molecule (usually a protein, carbohydrate or lipid) that is expressed on the surface of a cell, which is itself non-cancerous but for example promotes cancer cell growth or survival. For example, against immune cells to support cancer cells. Exemplary cells of this type include stromal cells and myeloid-derived suppressor cells (MDSCs). The tumor supporting antigen itself does not need to function in supporting tumor cells as long as the antigen is present on cells supporting the cancer cells.

The term "flexible polypeptide linker" or "linker" as used in the context of scFv refers to a peptide linker consisting of a glycine acid and/or a serine residue, such as used alone or in combination, which will be variable. The heavy chain region and the variable light chain region are joined together. In one embodiment, the flexible polypeptide linker is a Gly/Ser linker and comprises an amino acid sequence (Gly-Gly-Gly-Ser) _n (SEQ ID NO: 38), wherein n is equal to or greater than 1 A positive integer. For example, n=1, n=2, n=3, n=4, n=5, and n=6, n=7, n=8, n=9, and n=10. In one embodiment, the flexible polypeptide linker includes, but is not limited to, (Gly ₄ Ser) ₄ (SEQ ID NO: 27) or (Gly ₄ Ser) ₃ (SEQ ID NO: 28). In another embodiment, the linker comprises a plurality of repeats (Gly ₂ Ser), (GlySer) or (Gly ₃ Ser) (SEQ ID NO: 29). Linkers as described in WO2012/138475 are also included within the scope of the invention, which is incorporated herein by reference.

As used herein, a 5' capping (also known as RNA capping, RNA 7-methylguanosine capping or RNA m ⁷ G capping) is a modified guanine nucleotide that is added shortly after the start To the "front" or 5' end of the eukaryotic RNA. The 5' capping consists of a terminal group attached to the first transcribed nucleotide. Its presence is critical for ribosome recognition and protection from RNase action. The capping addition is combined with transcription and is carried out in an auxiliary transcriptional manner such that each affects each other. Shortly after initiation of transcription, the 5' end of the synthesized mRNA is bound by a capped synthetic complex associated with ribonucleic acid polymerase. This enzyme complex catalyzes the chemical reaction required for mRNA capping. The synthesis is carried out in the form of a multi-step biochemical reaction. The capping moiety can be modified to modulate the functionality of the mRNA, such as its translation stability or efficiency.

As used herein, "in vitro transcribed RNA" refers to RNA that has been synthesized in vitro, preferably mRNA. In general, in vitro transcribed RNA is produced from an in vitro transcription vector. In vitro transcription vectors contain templates for the production of RNA for in vitro transcription.

As used herein, "poly(A)" is a series of adenosine linked to mRNA by polyadenylation. In a preferred embodiment of the short-lived construct, the poly A is from 50 to 5000 (SEQ ID NO: 30), preferably greater than 64, more preferably greater than 100, and most preferably greater than 300 or 400. The poly(A) sequence can be chemically or enzymatically modified to modulate mRNA functionality, such as translational location, stability, or efficiency.

As used herein, "polyadenylation" refers to the covalent attachment of a polyadenylation moiety or a modified variant thereof to a messenger RNA molecule. In eukaryotes, most messenger RNA (mRNA) molecules are polyadenylated at the 3' end. The 3' poly(A) tail is a long sequence of adenine nucleotides (usually several hundred) added to the pre-mRNA via the action of an enzyme (polyadenylation polymerase). In higher eukaryotes, the poly(A) tail is added to a transcript containing a specific sequence (polyadenylation signal). The poly(A) tail and the proteins that bind to it help protect the mRNA from exonuclease degradation. Polyadenylation is important for transcriptional capping, mRNA export from the nucleus, and translation. Polyadenylation occurs in the nucleus immediately after transcription of the DNA into RNA, but can also occur later in the cytoplasm. Upon completion of transcription, the mRNA strand is cleaved by interaction with an endonuclease complex associated with RNA polymerase. The cleavage site is typically characterized by the presence of the base sequence AAUAAA near the cleavage site. After mRNA cleavage, adenosine residues are added to the free 3' end of the cleavage site.

As used herein, "transient" refers to a period of time in which a non-integrated transgenic gene is expressed for hours, days, or weeks, where the expression period is integrated into the genome or in a stable plastid replicon contained in a host cell. Less than the gene expression period.

As used herein, the term "treatment" refers to administration of one or more therapies (eg, one The or a plurality of therapeutic agents, such as the CAR of the invention, reduce or ameliorate the progression, severity and/or duration of the proliferative disorder, or ameliorate one or more symptoms of the proliferative disorder (preferably one or more discernible symptoms). In particular embodiments, the terms "treat", "treatment", and "treating" refer to at least one measurable physical parameter (such as tumor growth) that ameliorates a proliferative disorder without patient discrimination. . In other embodiments, the terms "treat", "treatment", and "treating" refer to a physiological manner by, for example, stabilizing a discernible symptom, by, for example, stabilizing physical parameters in a physiological manner. Inhibiting the progression of a proliferative disorder, or both. In other embodiments, the terms "treat", "treatment", and "treating" refer to reducing or stabilizing tumor size or number of cancer cells.

The term "signal transduction pathway" refers to the biochemical relationship between a variety of signal transduction molecules that function in transmitting a signal from one part of a cell to another part of the cell. The term "cell surface receptor" includes molecules and molecular complexes that are capable of receiving signals and transmitting signals across the cell membrane.

The term "individual" is intended to include living organisms (e.g., mammals, humans) that can elicit an immune response.

The term "substantially purified" cell refers to a cell that is substantially free of other cell types. Substantially purified cells also refer to cells that have been separated from other cell types that are normally associated with them in their naturally occurring state. In some cases, a substantially purified cell population system refers to a homogeneous cell population. In other instances, the term refers only to cells that have been separated from cells with which they are naturally associated in their natural state. In some aspects, the cells are cultured in vitro. In other aspects, the cells are not cultured in vitro.

The term "therapeutic" as used herein means treatment. Therapeutic effects are obtained by mitigating, inhibiting, alleviating or eradicating disease conditions.

The term "prevention" as used herein means prophylactic or protective treatment of a disease or condition.

In the context of the present invention, "tumor antigen" or "hyperproliferative disorder antigen" or "antigen associated with a hyperproliferative disorder" refers to an antigen that is common in a particular hyperproliferative disorder. In certain aspects, the hyperproliferative disorder antigen of the invention is derived from cancer, including but not limited to, primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin Lymphoma, Hodgkin's lymphoma, leukemia, uterine cancer, cervical cancer, bladder cancer, kidney cancer, and adenocarcinoma, such as breast cancer, prostate cancer (such as castration resistance or therapy-resistant prostate cancer or metastatic prostate cancer), Ovarian cancer, pancreatic cancer and the like, or plasma cell proliferative disorders such as asymptomatic myeloma (and flaccid myeloma or inert myeloma), unexplained gamma globulinemia (MGUS), Val Denver's macroglobulinemia, plasmacytoma (eg plasma cell cachexia, solitary myeloma, solitary plasmacytoma, extramedullary plasmacytoma and multiple plasmacytoma), systemic amyloid light chain Amyloidosis and POEMS syndrome (also known as Crohn-Fox syndrome, sorghum and PEP syndrome).

The term "transfection" or "transformation" or "transduction" refers to the process of transferring or introducing an exogenous nucleic acid into a host cell. A "transfection" or "transformation" or "transduction" cell is a cell that has been transfected, transformed or transduced with an exogenous nucleic acid. The cells include primary individual cells and their progeny.

The term "specifically binds" refers to an antibody or ligand that recognizes and binds to a homologous binding partner (eg, a stimulatory and/or costimulatory molecule present on a T cell) protein present in a sample, but the antibody or ligand Other molecules in the sample are not substantially identified or bound.

As used herein, "regulatable chimeric antigen receptor (RCAR)" refers to a group of polypeptides, typically two in the simplest embodiment, which, when administered in an immune effector cell, provides targeted targeting to the cell (usually cancer) The specificity of the cells) and the production of intracellular signals. In some embodiments, the RCAR comprises at least one extracellular antigen binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain"), in the case of a CAR molecule The lower cytoplasmic signaling domain comprises a stimulatory molecule derived from a protein as defined herein And/or a functional signaling domain of a co-stimulatory molecule. In some embodiments, the sets of polypeptides in the RCAR are not adjacent to each other, such as in different polypeptide chains. In some embodiments, the RCAR comprises a dimeric switch that can couple the polypeptides to each other in the presence of a dimeric molecule, for example, an antigen binding domain can be coupled to an intracellular signaling domain. In some embodiments, the RCAR is expressed in a cell (eg, an immune effector cell) as described herein, eg, a cell that exhibits RCAR (also referred to herein as "RCARX cells"). In one embodiment, the RCARX cells are T cells and are referred to as RCART cells. In one embodiment, the RCARX cells are NK cells and are referred to as RCARN cells. RCAR can provide RCAR-specific cells with specificity for target cells, usually cancer cells, and regulatable intracellular signal production or proliferation, which optimizes the immune response characteristics of cells expressing RCAR. In an embodiment, the RCAR cell is at least partially dependent on the antigen binding domain to provide specificity for a target cell comprising an antigen to which the antigen binding domain binds.

"Membrane anchor" or "membrane plug domain" as used herein, refers to a polypeptide or moiety (eg, myristyl) sufficient to anchor an extracellular or intracellular domain to the plasma membrane.

"Switching domain" when used herein, for example when referring to RCAR, refers to an entity associated with another switch domain in the presence of a dimeric molecule, typically a polypeptide-based entity. The association results in a functional coupling of a first entity coupled to (eg, fused to) the first switch domain to a second entity coupled to (eg, fused to) the second switch domain. The first and second switch domains are collectively referred to as a diplex switch. In an embodiment, the first and second switch domains are identical to each other, eg, they are polypeptides having the same primary amino acid sequence and are collectively referred to as homodimeric switches. In an embodiment, the first and second switch domains are different from each other, for example, they are polypeptides having different primary amino acid sequences and are collectively referred to as heterodimeric switches. In an embodiment, the switch is intracellular. In an embodiment, the switch is extracellular. In an embodiment, the switch domain is an entity based on a polypeptide (eg, based on FKBP or FRB) and the dimeric molecule is a small molecule, such as a rapalogue. In an embodiment, the switch domain is a polypeptide-based entity, such as a scFv that binds to a myc peptide, and the dimeric molecule is a polypeptide, a fragment thereof, or a plurality A multimer of a peptide, such as a myc ligand or a myc ligand that binds to one or more myc scFvs. In an embodiment, the switch domain is a polypeptide-based entity, such as the myc receptor, and the dimeric molecule is an antibody or fragment thereof, such as a myc antibody.

"Dimeric molecule" when used herein, for example when referring to RCAR, refers to a molecule that promotes association of a first switch domain with a second switch domain. In embodiments, the dimeric molecule is not naturally present in the individual or is present in a concentration that will result in significant dimerization. In an embodiment, the dimeric molecule is a small molecule such as rapamycin or rapamycin, such as RAD001.

The term "bioequivalent" refers to the amount of a non-reference compound (eg, RAD001) that produces an equivalent effect to a reference or reference amount of a reference compound (eg, RAD001). In one embodiment, the effect is as determined, for example, by inhibition of P70 S6 kinase, as assessed, for example, in an in vivo or in vitro assay, such as, for example, the assays described herein (eg, Boulay assay). The measured mTOR inhibition level or the measurement of the phosphorylated S6 content by the Western blot method. In one embodiment, the effect is to alter the ratio of PD-1 positive/PD-1 negative T cells, as measured by cell sorting. In one embodiment, the bioequivalent amount or dose of the mTOR inhibitor is an amount or dose of P70 S6 kinase inhibition that achieves the same level as the reference or reference amount of the reference compound. In one embodiment, the bioequivalent amount or dose of the mTOR inhibitor is an amount or dose that achieves a change in the ratio of PD-1 positive/PD-1 negative T cells at the same level as the reference or reference amount of the reference compound.

When used with, for example, an ectopic mTOR inhibitor (eg, RAD001 or rapamycin) or an mTOR inhibitor that catalyzes an mTOR inhibitor, the term "low immunopotentiating dose" refers to measurement as determined, for example, by inhibition of P70 S6 kinase activity, Partial (but not complete) dose of mTOR inhibitor that inhibits mTOR activity. Methods for assessing mTOR activity, for example, by inhibition of P70 S6 kinase are discussed herein. This dose is not sufficient to result in complete immunosuppression but is sufficient to enhance the immune response. In one embodiment, the low immunopotentiating dose of the mTOR inhibitor reduces the number of PD-1 positive immune effector cells (eg, T cells or NK cells) and/or PD-1 negative immunity. The number of inflammatory effector cells (such as T cells or NK cells) is increased, or the ratio of PD-1 negative immune effector cells (such as T cells or NK cells) / PD-1 positive immune effector cells (such as T cells or NK cells) is increased. high.

In one embodiment, the low immune booster dose of the mTOR inhibitor causes an increase in the number of native T cells. In one embodiment, the low immunopotentiating dose of the mTOR inhibitor causes one or more of the following: for example, an increase in one or more of the following markers on memory T cells such as memory T cell precursor cells: CD62L ^High , CD127 ^high , CD27 ⁺ and BCL2; for example, a decrease in KLRG1 expression on memory T cells such as memory T cell precursor cells; and an increase in the number of memory T cell precursor cells, for example having any of the following characteristics Or combined cells: ^high increase in CD62L, increase in CD127 ^high, increase in CD27 ⁺ , decrease in KLRG1, and increase in BCL2; wherein any of the above-described changes, for example, occur at least briefly, as compared to, for example, an untreated individual.

As used herein, "refractory" refers to a disease that does not respond to treatment, such as cancer. In an embodiment, the refractory cancer may be resistant to treatment prior to or at the beginning of treatment. In other embodiments, the refractory cancer may become resistant during treatment. Refractory cancer is also known as resistant cancer.

As used herein, "relapsed" or "relapse" refers to a disease (eg, cancer) or a disease such as cancer after an improvement or reaction period, such as, for example, prior treatment of a therapy such as cancer therapy. The signs and symptoms are reproduced. For example, the reaction period can include the level of cancer cells falling below a certain threshold, such as 20%, 1%, 10%, 5%, 4%, 3%, 2%, or 1% or less. Reproduction may include raising the level of cancer cells above a certain threshold, such as 20%, 1%, 10%, 5%, 4%, 3%, 2%, or 1% or more.

Scope: Throughout the present invention, various aspects of the present invention may be in a range format Now. It should be understood that the description of the range format is merely for convenience and brevity and should not be construed as a limitation of the scope of the invention. Accordingly, the description of a range should be considered as a particular disclosure of all possible sub-ranges and individual values within the scope. For example, a range description such as 1 to 6 should be considered to have specifically disclosed sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, and the like, and individual within the range Numbers, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a conformance range such as 95% to 99% includes 95%, 96%, 97%, 98%, or 99% consistency, and includes sub-ranges, such as 96% to 99%, 96% to 98. %, 96% to 97%, 97% to 99%, 97% to 98%, and 98% to 99% consistency. This applies regardless of the width of the range.

description

Provided herein are subject compositions and methods of use for treating diseases such as cancer using cells that exhibit BCMA chimeric antigen receptor (CAR), such as CART-BCMA.

In one aspect, the invention provides a plurality of chimeric antigen receptors (CARs) comprising an antibody or antibody fragment engineered to enhance binding to a BCMA protein. In one aspect, the invention provides cells engineered to express CAR (eg, immune effector cells, such as T cells or NK cells), wherein CART cells ("CART") or CAR NK cells exhibit anti-tumor properties. In one aspect, the cells are transformed with CAR and the CAR is expressed on the cell surface. In some embodiments, a cell (eg, an immune effector cell, such as a T cell or an NK cell) is transduced with a viral vector encoding a CAR. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is a lentiviral vector. In some such embodiments, the cells can stably express CAR. In another embodiment, a cell (eg, an immune effector cell, such as a T cell or NK cell) is transfected with a nucleic acid encoding a CAR, such as mRNA, cDNA, DNA. In some such embodiments, the cells can transiently express CAR.

In one aspect, the anti-BCMA antigen binding portion of CAR is a scFv antibody fragment. In one aspect, such antibody fragments are functional because they retain equivalent binding affinity, i.e., they bind to the same antigen as the IgG antibody from which they are derived, with comparable efficacy. In other embodiments, the antibody fragment has a lower binding affinity, for example, it binds to the same antigen with a lower binding affinity than the antibody from which it is derived, but is functional because it provides the biological response described herein. In one embodiment, CAR molecule comprising a target antigen of 10 ^-4 M to 10 ^-8 M, for example, 10 ^-5 M to 10 ^-7 M, 10 ^-6 M, or in conjunction with, for example, 10 ^-7 M of the affinity of KD Antibody fragment. In one embodiment, the antibody fragment has a binding affinity that is at least five, ten, 20, 30, 50, 100 or 1,000 fold lower than a reference antibody, such as an antibody described herein.

In one aspect, such antibody fragments function because they provide, but are not limited to, biological reactions as will be appreciated by those skilled in the art: activating immune responses, inhibiting signal transduction from their target antigens Origin, inhibition of kinase activity and its similar biological response. In one aspect, the anti-BCMA antigen binding domain of CAR is a scFv antibody fragment that is humanized compared to the murine sequence from which the scFv is derived. In one embodiment, the anti-BCMA antigen binding domain is a human anti-BCMA antigen binding domain. In one embodiment, the anti-BCMA antigen binding domain is a humanized anti-BCMA antigen binding domain.

In some aspects, the antibodies of the invention are incorporated into a chimeric antigen receptor (CAR). In one aspect, the CAR comprises SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45 SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO : 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145 , SEQ ID NO: 146, SEQ BCMA binding domain of the sequence of ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 265 or SEQ ID NO: 266. In one aspect, the scFv domain is human. In another aspect, the scFv domain is PCT Publication No. WO 2012/163805, U.S. Patent No. 7,083,785, EP Patent No. 1975231 B1, or PCT Publication No. WO 13/154760 (each of which is incorporated by reference in its entirety. The manner in which the humanized variants of the scFv domain of the antibody or antibody fragment described herein are incorporated herein, which disclose a murine-derived antibody or scFv fragment that specifically binds to human BCMA. Humanization of such mouse antibodies and/or scFvs may be required for clinical settings in which mouse-specific residues may be treated with CART-BCMA, such as immune effector cells transduced with anti-BCMA CAR constructs (eg, Human anti-mouse antigen (HAMA) responses are induced in patients treated with T cells or NK cells.

In one aspect, the anti-BCMA binding domain (e.g., human or humanized scFv) of a CAR of the invention is partially encoded by a sequence that has been shown to be codon-optimized in a mammalian cell. In one aspect, the entire CAR construct of the invention has been encoded by a whole sequence that has been shown to be codon-optimized in a mammalian cell. Codon optimization refers to the discovery that synonymous codons (i.e., codons encoding the same amino acid) are biased in the encoded DNA in different species. Such codon degeneracy allows the same polypeptide to be encoded by a variety of nucleotide sequences. A variety of codon optimisation methods are known in the art and include, for example, the methods disclosed in at least U.S. Patent Nos. 5,786,464 and 6,114,148.

In one aspect, the human anti-BCMA binding domain comprises the scFv portion provided in SEQ ID NO:39. In one aspect, the human anti-BCMA CAR comprises the scFv portion provided in SEQ ID NO:40. In one aspect, the human anti-BCMA binding domain comprises the scFv portion provided in SEQ ID NO:41. In one aspect, the human anti-BCMA binding domain comprises the scFv portion provided in SEQ ID NO:42. In one aspect, the human anti-BCMA binding domain comprises the one provided in SEQ ID NO:43 The scFv part. In one aspect, the human anti-BCMA binding domain comprises the scFv portion provided in SEQ ID NO:44. In one aspect, the human anti-BCMA binding domain comprises the scFv portion provided in SEQ ID NO:45. In one aspect, the human anti-BCMA binding domain comprises the scFv portion provided in SEQ ID NO:46. In one aspect, the human anti-BCMA binding domain comprises the scFv portion provided in SEQ ID NO:47. In one aspect, the human anti-BCMA binding domain comprises the scFv portion provided in SEQ ID NO:48. In one aspect, the human anti-BCMA binding domain comprises the scFv portion provided in SEQ ID NO:49. In one aspect, the human anti-BCMA binding domain comprises the scFv portion provided in SEQ ID NO:50. In one aspect, the human anti-BCMA binding domain comprises the scFv portion provided in SEQ ID NO:51. In one aspect, the human anti-BCMA binding domain comprises the scFv portion provided in SEQ ID NO:52. In one aspect, the human anti-BCMA binding domain comprises the scFv portion provided in SEQ ID NO:53. In one aspect, the human anti-BCMA binding domain comprises the scFv portion provided in SEQ ID NO: 129. In one aspect, the human anti-BCMA binding domain comprises the scFv portion provided in SEQ ID NO:130. In one aspect, the human anti-BCMA CAR comprises the scFv portion provided in SEQ ID NO:131. In one aspect, the human anti-BCMA binding domain comprises the scFv portion provided in SEQ ID NO:132. In one aspect, the human anti-BCMA binding domain comprises the scFv portion provided in SEQ ID NO:133. In one aspect, the human anti-BCMA binding domain comprises the scFv portion provided in SEQ ID NO: 134. In one aspect, the human anti-BCMA binding domain comprises the scFv portion provided in SEQ ID NO:135. In one aspect, the human anti-BCMA binding domain comprises the scFv portion provided in SEQ ID NO: 136. In one aspect, the human anti-BCMA binding domain comprises the scFv portion provided in SEQ ID NO:137. In one aspect, the human anti-BCMA binding domain comprises the scFv portion provided in SEQ ID NO:138. In one aspect, the human anti-BCMA binding domain comprises SEQ ID NO:139 The scFv part provided. In one aspect, the human anti-BCMA binding domain comprises the scFv portion provided in SEQ ID NO:140. In one aspect, the human anti-BCMA binding domain comprises the scFv portion provided in SEQ ID NO:141. In one aspect, the human anti-BCMA CAR comprises the scFv portion provided in SEQ ID NO:142. In one aspect, the human anti-BCMA CAR comprises the scFv portion provided in SEQ ID NO: 143. In one aspect, the human anti-BCMA CAR comprises the scFv portion provided in SEQ ID NO:144. In one aspect, the human anti-BCMA CAR comprises the scFv portion provided in SEQ ID NO:145. In one aspect, the human anti-BCMA CAR comprises the scFv portion provided in SEQ ID NO: 146. In one aspect, the human anti-BCMA CAR comprises the scFv portion provided in SEQ ID NO: 147. In one aspect, the human anti-BCMA CAR comprises the scFv portion provided in SEQ ID NO: 148. In one aspect, the human anti-BCMA CAR comprises the scFv portion provided in SEQ ID NO:149. In one aspect, the humanized anti-BCMA binding domain comprises the scFv portion provided in SEQ ID NO:255. In one aspect, the humanized anti-BCMA CAR comprises the scFv portion provided in SEQ ID NO:257.

In one aspect, the human anti-BCMA CAR comprises the scFv portion provided in SEQ ID NO:263. In one aspect, the human anti-BCMA CAR comprises the scFv portion provided in SEQ ID NO:264. In one aspect, the human anti-BCMA CAR comprises the scFv portion provided in SEQ ID NO:265. In one aspect, the human anti-BCMA CAR comprises the scFv portion provided in SEQ ID NO:266.

In one aspect, the CAR of the invention combines an antigen binding domain of an antibody with an intracellular signaling molecule. For example, in some aspects, intracellular signaling molecules include, but are not limited to, CD3-ξ chains, 4-1BB and CD28 signaling modules, and combinations thereof. In one aspect, the antigen binding domain binds to BCMA. In one aspect, the BCMA CAR comprises a CAR selected from the sequence provided in one or more of the following: SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO : 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218 SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, and SEQ ID NO: 233. In one aspect, the BCMA CAR comprises the sequence provided in SEQ ID NO:99. In one aspect, the BCMA CAR comprises the sequence provided in SEQ ID NO:100. In one aspect, the BCMA CAR comprises the sequence provided in SEQ ID NO:101. In one aspect, BCMA CAR comprises the sequence provided in SEQ ID NO:102. In one aspect, the BCMA CAR comprises the sequence provided in SEQ ID NO:103. In one aspect, BCMA CAR comprises the sequence provided in SEQ ID NO:104. In one aspect, BCMA CAR comprises the sequence provided in SEQ ID NO:105. In one aspect, BCMA CAR comprises the sequence provided in SEQ ID NO:106. In one aspect, the BCMA CAR comprises the sequence provided in SEQ ID NO:107. In one aspect, the BCMA CAR comprises the sequence provided in SEQ ID NO:108. In one aspect, BCMA CAR comprises the sequence provided in SEQ ID NO:109. In one aspect, BCMA CAR comprises the sequence provided in SEQ ID NO:110. In one aspect, BCMA CAR comprises the sequence provided in SEQ ID NO:111. In one aspect, the BCMA CAR comprises the sequence provided in SEQ ID NO:112. In one aspect, BCMA CAR comprises the sequence provided in SEQ ID NO:213. In one aspect, BCMA CAR comprises the sequence provided in SEQ ID NO:214. In one aspect, BCMA CAR comprises the sequence provided in SEQ ID NO:215. In one aspect, BCMA CAR contains SEQ ID NO: The sequence provided in 216. In one aspect, BCMA CAR comprises the sequence provided in SEQ ID NO:217. In one aspect, BCMA CAR comprises the sequence provided in SEQ ID NO:218. In one aspect, BCMA CAR comprises the sequence provided in SEQ ID NO:219. In one aspect, BCMA CAR comprises the sequence provided in SEQ ID NO:220. In one aspect, BCMA CAR comprises the sequence provided in SEQ ID NO:221. In one aspect, BCMA CAR comprises the sequence provided in SEQ ID NO:222. In one aspect, BCMA CAR comprises the sequence provided in SEQ ID NO:223. In one aspect, BCMA CAR comprises the sequence provided in SEQ ID NO:224. In one aspect, BCMA CAR comprises the sequence provided in SEQ ID NO:225. In one aspect, BCMA CAR comprises the sequence provided in SEQ ID NO:226. In one aspect, BCMA CAR comprises the sequence provided in SEQ ID NO:227. In one aspect, BCMA CAR comprises the sequence provided in SEQ ID NO:228. In one aspect, BCMA CAR comprises the sequence provided in SEQ ID NO:229. In one aspect, BCMA CAR comprises the sequence provided in SEQ ID NO:230. In one aspect, BCMA CAR comprises the sequence provided in SEQ ID NO:231. In one aspect, BCMA CAR comprises the sequence provided in SEQ ID NO:232. In one aspect, BCMA CAR comprises the sequence provided in SEQ ID NO:233.

Furthermore, the present invention provides BCMA CAR compositions and their use in the treatment of a medicament or method for treating cancer or any malignant or autoimmune disease involving cells or tissues of BCMA in other diseases.

In one aspect, the CAR of the present invention can be used to eradicate normal cells expressing BCMA and is therefore suitable for use as a cell-adjusting therapy prior to cell transplantation. In one aspect, the normal cells expressing BCMA are normal stem cells that express BCMA and the cells are transplanted into stem cell transplants.

In one aspect, the invention provides engineered to express a chimeric antigen receptor (CAR) cells (eg, T cells or NK cells) in which CART cells ("CART") or CAR NK cells exhibit anti-tumor properties. A preferred antigen is BCMA. In one aspect, the antigen binding domain of CAR comprises a human anti-BCMA antibody fragment or a partially humanized anti-BCMA antibody fragment. In one aspect, the antigen binding domain of CAR comprises a human anti-BCMA antibody fragment comprising a scFv or a partially humanized anti-BCMA antibody fragment. Accordingly, the invention provides a BCMA-CAR comprising a humanized anti-BCMA binding domain and engineered into a cell, such as a T cell or NK cell; and methods of use thereof for adoptive therapy.

In one aspect, the BCMA-CAR comprises at least one intracellular domain selected from the group consisting of a CD137 (4-1BB) signaling domain, a CD28 signaling domain, a CD3ζ signaling domain, and any combination thereof. In one aspect, the BCMA-CAR comprises at least one intracellular signaling domain from one or more costimulatory molecules other than CD137 (4-1BB) or CD28.

Chimeric antigen receptor (CAR)

The invention provides a CAR (eg, a CAR polypeptide) comprising an anti-BCMA binding domain (eg, a human or humanized anti-BCMA binding domain as described herein), a transmembrane domain, and an intracellular signaling domain, And wherein the anti-BCMA binding domain comprises the heavy chain complementarity determining region 1 (HC CDR1) and the heavy chain complementarity determining region 2 of any anti-BMCA heavy chain binding domain amino acid sequence listed in Table 1 or 16 ( HC CDR2) and heavy chain complementarity determining region 3 (HC CDR3). The anti-BCMA binding domain of CAR may further comprise the light chain complementarity determining region 1 (LC CDR1), light chain complementarity determining region 2 of any anti-BMCA light chain binding domain amino acid sequence listed in Table 1 or 16. (LC CDR2) and light chain complementarity determining region 3 (LC CDR3).

The invention also provides a nucleic acid molecule encoding a CAR as described herein, for example, encoding an anti-BCMA binding domain (eg, a human or humanized BCMA binding domain as described herein), a transmembrane domain, and an intracellular signal. The CAR of the conduction domain, and wherein the anti- The BCMA binding domain comprises the heavy chain complementarity determining region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2) and heavy of any anti-BCMA heavy chain binding domain amino acid sequence listed in Table 1 or 16 Chain complementarity determining region 3 (HC CDR3). In one embodiment, the encoded anti-BCMA binding domain of CAR may further comprise the light chain complementarity determining region 1 of any anti-BMCA light chain binding domain amino acid sequence listed in Table 1 or 16 (LC) CDR1), light chain complementarity determining region 2 (LC CDR2) and light chain complementarity determining region 3 (LC CDR3).

In a particular aspect, the CAR construct of the invention comprises a strain selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44 SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO : 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144 SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 265, and SEQ ID NO: a scFv domain of a population consisting of 266, wherein the scFv can be preceded by a preamble as provided in SEQ ID NO: 1, and then optionally as SEQ ID NO: 2 or SEQ ID NO: 3 or the hinge provided in SEQ ID NO: 4 or SEQ ID NO: 5 A sequence, such as the transmembrane region provided in SEQ ID NO: 6, an intracellular signaling domain comprising SEQ ID NO: 7 or SEQ ID NO: 8, and a CD3 comprising SEQ ID NO: 9 or SEQ ID NO: A sequence wherein the domains are adjacent and in the same reading frame to form a single fusion protein. The invention also includes a nucleotide sequence encoding a polypeptide of each scFv fragment selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42 SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 265 and SEQ ID NO:266.

The invention also includes a nucleotide sequence encoding a polypeptide of each scFv fragment selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42 SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO : 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142 SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 265 and SEQ ID NO: 266, and each of SEQ ID NO: 1, 2 and 6-9, plus the encoded BCMA CAR fusion protein of the invention.

In one aspect, an exemplary BCMA CAR construct comprises a pre-sequence, an extracellular antigen binding domain, a hinge, a transmembrane domain, and an intracellular stimulatory domain, as appropriate. In one aspect, the exemplary BCMA CAR construct contains pre-existing conditions as appropriate Sequences, extracellular antigen binding domains, hinges, transmembrane domains, intracellular co-stimulatory domains, and intracellular stimulatory domains. Specific BCMA CAR constructs comprising a human scFv domain of the invention are provided as SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44. SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO:53, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135,SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, and SEQ ID NO: 149. The full-length CAR sequence is also provided herein as SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44 as shown in Table 1. SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO : 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144 SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, and SEQ ID NO: 149.

An exemplary leader sequence is provided as SEQ ID NO: 1. An exemplary hinge/spacer sequence is provided as SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5. An exemplary transmembrane domain sequence is provided as SEQ ID NO: 6. An exemplary sequence of the intracellular signaling domain of the 4-1BB protein is provided as SEQ ID NO:7. CD27 An exemplary sequence of an internal signaling domain is provided as SEQ ID NO:8. An exemplary CD3ζ domain sequence is provided as SEQ ID NO: 9 or SEQ ID NO: 10.

In one aspect, the invention encompasses a recombinant nucleic acid construct comprising a nucleic acid molecule encoding a CAR, wherein the nucleic acid molecule comprises a nucleic acid sequence encoding, for example, an anti-BCMA binding domain as described herein, which encodes intracellular signaling The nucleic acid sequences of the domains are contiguous and in the same reading frame. In one aspect, the anti-BCMA binding domain is selected from one or more of the group consisting of: SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO :58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66 SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, and SEQ ID NO: 170. In one aspect, the anti-BCMA binding domain comprises SEQ ID NO:54. In one aspect, the anti-BCMA binding domain comprises SEQ ID NO:55. In one aspect, the anti-BCMA binding domain comprises SEQ ID NO:56. In one aspect, the anti-BCMA binding domain comprises SEQ ID NO:57. In one aspect, the anti-BCMA binding domain comprises SEQ ID NO:58. In one aspect, the anti-BCMA binding domain comprises SEQ ID NO:59. In one aspect, the anti-BCMA binding domain comprises SEQ ID NO:60. In one aspect, the anti-BCMA binding domain comprises SEQ ID NO:61. In one aspect, the anti-BCMA binding domain comprises SEQ ID NO:62. In one aspect, the anti-BCMA binding domain comprises SEQ ID NO:63. In one aspect, the anti-BCMA binding domain comprises SEQ ID NO:64. In one aspect, the anti-BCMA binding domain comprises SEQ ID NO: 65. In one aspect, the anti-BCMA binding domain comprises SEQ ID NO:66. In one aspect, the anti-BCMA binding domain comprises SEQ ID NO:67. In one aspect, the anti-BCMA binding domain comprises SEQ ID NO:68. In one aspect, the anti-BCMA binding domain comprises SEQ ID NO:150. In one aspect, the anti-BCMA binding domain comprises SEQ ID NO:151. In one aspect, the anti-BCMA binding domain comprises SEQ ID NO:152. In one aspect, the anti-BCMA binding domain comprises SEQ ID NO:153. In one aspect, the anti-BCMA binding domain comprises SEQ ID NO:154. In one aspect, the anti-BCMA binding domain comprises SEQ ID NO:155. In one aspect, the anti-BCMA binding domain comprises SEQ ID NO:156. In one aspect, the anti-BCMA binding domain comprises SEQ ID NO:157. In one aspect, the anti-BCMA binding domain comprises SEQ ID NO:158. In one aspect, the anti-BCMA binding domain comprises SEQ ID NO: 159. In one aspect, the anti-BCMA binding domain comprises SEQ ID NO:160. In one aspect, the anti-BCMA binding domain comprises SEQ ID NO:161. In one aspect, the anti-BCMA binding domain comprises SEQ ID NO:162. In one aspect, the anti-BCMA binding domain comprises SEQ ID NO:163. In one aspect, the anti-BCMA binding domain comprises SEQ ID NO:164. In one aspect, the anti-BCMA binding domain comprises SEQ ID NO:165. In one aspect, the anti-BCMA binding domain comprises SEQ ID NO:166. In one aspect, the anti-BCMA binding domain comprises SEQ ID NO:167. In one aspect, the anti-BCMA binding domain comprises SEQ ID NO:168. In one aspect, the anti-BCMA binding domain comprises SEQ ID NO:169. In one aspect, the anti-BCMA binding domain comprises SEQ ID NO:170.

In one aspect, the invention encompasses a recombinant nucleic acid construct comprising a nucleic acid molecule encoding a CAR, wherein the nucleic acid molecule comprises a nucleic acid sequence encoding an anti-BCMA binding domain selected from one or more of the following: SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, and SEQ ID NO: 170, eg, wherein the sequence is adjacent to the nucleic acid sequence encoding the intracellular signaling domain and is read in the same In the frame. One exemplary intracellular signaling domain useful in a CAR includes, but is not limited to, one or more intracellular signaling domains such as CD3-ξ, CD28, 4-1BB, and the like. In some cases, the CAR can comprise any combination of CD3-ξ, CD28, 4-1BB, and the like. In one aspect, the nucleic acid sequence of the CAR construct of the invention is selected from the group consisting of SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, one or more of SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252, SEQ ID NO: 253 or SEQ ID NO: 254. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO:114. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO:115. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO:116. In one aspect, the nucleic acid sequence of the CAR construct Is SEQ ID NO:117. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO:118. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO: 119. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO:120. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO:121. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO:122. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO:123. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO:124. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO:125. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO: 126. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO:127. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO:128. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO:234. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO:235. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO:236. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO:237. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO:238. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO:239. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO:240. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO:241. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO: 242. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO:243. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO:244. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO:245. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO:246. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO:247. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO: 248. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO:249. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO:250. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO:251. In one aspect, CAR The nucleic acid sequence of the construct is SEQ ID NO:252. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO:253. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO:254.

The nucleic acid sequence encoding the desired molecule can be obtained using recombinant methods known in the art, such as by using standard techniques, from a cell screening library of expression genes, from genes known to include genes, or from cells and tissues containing genes. Direct separation. Alternatively, the relevant nucleic acid can be produced synthetically rather than colonized.

The invention encompasses retroviral and lentiviral vector constructs that express a CAR that can be directly transduced into a cell.

The invention also encompasses RNA constructs that can be directly transfected into cells. The method of generating mRNA for transfection involves the use of a specially designed primer in vitro transcription (IVT) template followed by the addition of poly A to generate 3' and 5' untranslated sequences ("UTR"), 5' capping and/or Or an internal ribosome entry site (IRES), a nucleic acid to be expressed, and a construct of a poly A tail (SEQ ID NO: 35) of 50-2000 bases in length. The RNA produced can be efficiently transfected into different types of cells. In one embodiment, the template comprises a sequence of CARs. In one embodiment, the RNA CAR vector is transduced into cells, such as T cells or NK cells, by electroporation.

Antigen binding domain

The CAR of the invention comprises a target specific binding domain. Partial selection depends on the type and number of ligands that define the surface of the target cell. For example, an antigen binding domain can be selected to recognize an antigen that acts as a cell surface marker on a target cell associated with a particular disease condition.

In one aspect, a CAR-mediated T cell response can be directed against a related antigen by engineering an antigen binding domain that specifically binds the desired antigen into the CAR.

In one aspect, the CAR of the invention comprises a binding domain that specifically binds to BCMA. In one aspect, the CAR of the invention comprises an antigen binding domain that specifically binds to human BCMA.

The antigen binding domain can be any protein that binds to an antigen, including but not limited to monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, and functional fragments thereof, including but not limited to Domain antibodies, such as the heavy chain variable domain (VH), the light chain variable domain (VL), and the variable domain of the camelid-derived nanobody (VHH); and combinations known in the art The backbone acts to act as an antigen binding domain, such as a recombinant fibronectin domain and analogs thereof. In some cases, the antigen binding domain is preferably derived from the same species that will ultimately use the CAR. For example, for use in humans, the antigen binding domain of CAR preferably comprises a human or humanized residue for the antigen binding domain of an antibody or antibody fragment.

Thus, in one aspect, the antigen binding domain comprises a human or humanized antibody or antibody fragment. In one embodiment, the human anti-BCMA binding domain comprises one or more (eg, all three) light chain complementarity determining regions 1 (LC CDR1), light chain complementary, of the human anti-BCMA binding domain described herein. Determining region 2 (LC CDR2) and light chain complementarity determining region 3 (LC CDR3), and/or one or more (eg, all three) heavy chain complementarity determining regions 1 of the human anti-BCMA binding domain described herein (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2) and heavy chain complementarity determining region 3 (HC CDR3), for example comprising one or more, for example all three LC CDRs and one or more, for example all three Human anti-BCMA binding domain of HC CDRs. In one embodiment, the human anti-BCMA binding domain comprises one or more (eg, all three) heavy chain complementarity determining regions 1 (HC CDR1), heavy chain complementary, of the human anti-BCMA binding domain described herein. Defining region 2 (HC CDR2) and heavy chain complementarity determining region 3 (HC CDR3), for example, the human anti-BCMA binding domain has two variable heavy chain regions, each variable heavy region comprising the HC CDR1 described herein , HC CDR2 and HC CDR3. In one embodiment, the human anti-BCMA binding domain comprises a human light chain variable region (eg, in Table 1) as described herein and/or a human heavy chain variable region (eg, in Table 1) as described herein. In one embodiment, the human anti-BCMA binding domain comprises a human heavy chain variable region as described herein (eg, in Table 1), eg, at least two human heavy chain variants described herein are variable Zone (for example, in Table 1). In one embodiment, the anti-BCMA binding domain is a scFv comprising the light and heavy chains of the amino acid sequence of Table 1. In one embodiment, the anti-BCMA binding domain (eg, scFv) comprises: at least one, two or three modifications (eg, substitutions) comprising an amino acid sequence having a light chain variable region as provided in Table 1. , for example, a conservative substitution) but no more than 30, 20 or 10 modifications (eg, substitutions, such as conservative substitutions) of the amino acid sequence or sequences having 95% to 99% identity to the amino acid sequence of Table 1 a light chain variable region; and/or comprising at least one, two or three modifications (eg, substitutions, such as conservative substitutions) having no more than 30 amino acid sequences to the heavy chain variable regions provided in Table 1. The amino acid sequence of 20, 10 or 10 modifications (e.g., substitutions, such as conservative substitutions) or the heavy chain variable region of the sequence having 95% to 99% identity to the amino acid sequence of Table 1.

In one embodiment, the human anti-BCMA binding domain comprises selected from the group consisting of SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO: 44. SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO:53, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135,SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144. A sequence of a population consisting of SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, and SEQ ID NO: 149, or a sequence having 95% to 99% identity thereto. In one embodiment, the nucleic acid sequence encoding the human anti-BCMA binding domain comprises selected from the group consisting of SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, a sequence of a population consisting of SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, and SEQ ID NO:170, or which is 95%-99% identical thereto Sequence of sex. In one embodiment, the human anti-BCMA binding domain is a scFv and the light chain variable region comprising an amino acid sequence (eg, in Table 1) described herein is via a linker (eg, a linker as described herein) Attached to the heavy chain variable region comprising an amino acid sequence as described herein (e.g., in Table 1). In one embodiment, the human anti-BCMA binding domain comprises a (Gly ₄ -Ser) n linker, wherein n is 1, 2, 3, 4, 5 or 6, preferably 3 or 4 (SEQ ID NO: 26). The light chain variable region and the heavy chain variable region of the scFv can, for example, be in any of the following orientations: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain Variable zone. In one aspect, the antigen binding domain portion comprises one or more selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, and SEQ ID NO: 149. In one aspect, the CAR is selected from one or more selected from the group consisting of SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, and SEQ ID NO: 233.

In one embodiment, the anti-BCMA binding domain comprises a light chain variable region as described herein (eg, in Table 16) and/or a heavy chain variable region (eg, in Table 16) as described herein. In one embodiment, the encoded humanized anti-BCMA binding domain comprises the light chain variable region provided in SEQ ID NO:259, SEQ ID NO:260, SEQ ID NO:261, SEQ ID NO:262 And/or the heavy chain variable region provided in SEQ ID NO: 255, SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258. In one embodiment, the encoded anti-BCMA binding domain is a scFv comprising the light and heavy chains of the amino acid sequence of Table 16. In one embodiment, the human or humanized anti-BCMA binding domain (eg, scFv) comprises: comprising the pair of SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 261, SEQ ID NO: Providing at least one, two or three modifications (eg, substitutions, eg, conservative substitutions) of the amino acid sequence of the light chain variable region but no more than 30, 20 or 10 modifications (eg, substitutions, such as conservative substitutions) An amino acid sequence, or a light chain variable region thereof having a sequence with 95%-99% identity; and/or comprising a pair of SEQ ID NO: 255, SEQ ID NO: 256, SEQ ID NO: 257, SEQ At least one, two or three modifications (eg, substitutions, eg, conservative substitutions) of the amino acid sequence of the heavy chain variable region provided in ID NO: 258 but no more than 30, 20 or 10 modifications (eg, Substituting, for example, a conservatively substituted amino acid sequence, or a heavy chain variable region thereof having a sequence that is 95% to 99% identical. In one embodiment, the encoded anti-BCMA binding domain comprises a (Gly ₄ -Ser) n linker, wherein n is 1, 2, 3, 4, 5 or 6, preferably 3 or 4 (SEQ ID NO: 26). The light chain variable region and the heavy chain variable region of the scFv can, for example, be in any of the following orientations: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain Variable zone.

In one embodiment, the human anti-BCMA binding domain comprises or consists of a sequence selected from the group consisting of SEQ ID NO:263, SEQ ID NO:264, SEQ ID NO:265, and SEQ ID NO:266 -99% sequence of consistency.

In some aspects, a non-human antibody is humanized, wherein a particular sequence or region of the antibody is modified to increase the similarity to antibodies naturally produced in humans or fragments thereof. In one aspect, the antigen binding domain is humanized.

Humanized antibodies can be produced using a variety of techniques known in the art, including, but not limited to, CDR grafting (see, for example, European Patent No. EP 239,400; International Publication No. WO 91/09967; and U.S. Patent No. 5,225,539 U.S. Patent Nos. 5,530,101 and 5,585,089, each incorporated herein by reference in its entirety, in the entire entire entire entire entire entire entire entire entire entire entire entire entire- (4/5): 489-498; Studnicka et al., 1994, Protein Engineering, 7(6): 805-814; and Roguska et al., 1994, PNAS, 91: 969-973, each of which is incorporated by reference in its entirety. </ RTI> </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; Application Publication No. US2005/0048617, U.S. Patent No. 6,407,213, U.S. Patent No. 5,766,886, International Publication No. WO 9317105; Tan et al., J. Immunol., 169: 1119-25 (2002); Caldas et al. , Protein Eng., 13(5): 353 -60 (2000); Morea et al, Methods, 20(3): 267-79 (2000); Baca et al, J. Biol. Chem., 272(16): 10678-84 (1997); Roguska et al. , Protein Eng., 9(10): 895-904 (1996); Couto et al., Cancer Res., 55 (23 Supp): 5973s- 5977s (1995); Couto et al, Cancer Res., 55(8): 1717-22 (1995); Sandhu JS, Gene, 150(2): 409-10 (1994); and Pedersen et al., J. Mol .Biol., 235(3): 959-73 (1994), each of which is incorporated herein by reference in its entirety. Typically, framework residues in the framework regions will be altered by substitution of corresponding residues from the CDR donor antibody, for example to increase antigen binding. Such framework substitutions, such as conservative substitutions, are identified by methods well known in the art, for example by modeling the interaction of CDRs with framework residues to identify framework residues important for antigen binding and to sequence comparisons. Unusual framework residues are identified at specific positions (see, for example, Queen et al., U.S. Patent No. 5,585,089; and Riechmann et al., 1988, Nature, 332: 323, which is incorporated herein by reference in its entirety).

One or more amino acid residues from a non-human source are retained in the humanized antibody or antibody fragment. Such non-human amino acid residues are often referred to as "input" residues, which are typically taken from the "input" variable domain. As provided herein, a humanized antibody or antibody fragment comprises one or more CDRs from a non-human immunoglobulin molecule, and a framework region in which the amino acid residues that make up the framework are fully or largely derived from the human germline. A number of techniques for humanizing antibodies or antibody fragments are well known in the art and can be substantially according to Winter and colleagues (Jones et al, Nature, 321 :522-525 (1986); Riechmann et al, Nature, 332: 323-327 (1988); Verhoeyen et al, Science, 239: 1534-1536 (1988)), by replacing the corresponding sequence of a human antibody with a rodent CDR or CDR sequence, ie CDR grafting (EP 239,400; PCT Publication No. WO 91/09967; and U.S. Patent Nos. 4,816,567; 6,331,415; 5,225,539; 5,530,101; 5,585,089; 6, 548, 640, the contents of each of . In such humanized antibodies and antibody fragments, substantially less than the entire human variable domain has been replaced by the corresponding sequence of a non-human substance. Humanized antibodies are typically human antibodies in which some of the CDR residues and possibly some framework (FR) residues are substituted with residues from analogous sites in rodent antibodies. Humanization of antibodies and antibody fragments can also be borrowed Renovated by inlays or surfaces (EP 592,106; EP 519,596; Padlan, 1991, Molecular Immunology, 28(4/5): 489-498; Studnicka et al, Protein Engineering, 7(6): 805-814 (1994); And Roguska et al., PNAS, 91: 969-973 (1994)) or chain reorganization (U.S. Patent No. 5,565,332), the disclosure of which is incorporated herein by reference.

The selection of human variable domains (light and heavy) that can be used to make humanized antibodies will reduce antigenicity. The variable domain sequences of rodent antibodies are screened against the entire library of known human variable domain sequences according to the so-called "best fit" method. The human sequence closest to the rodent is then accepted as a human framework (FR) for humanized antibodies (Sims et al, J. Immunol., 151: 2296 (1993); Chothia et al, J. Mol. Biol., 196:901 (1987), the contents of which are incorporated herein by reference in its entirety. Another method uses a specific framework derived from the common sequence of all human antibodies of a particular subset of light or heavy chains. The same framework can be used for several different humanized antibodies (see, for example, Nicholson et al. Mol. Immun. 34 (16-17): 1157-1165 (1997); Carter et al, Proc. Natl. Acad. Sci. USA, 89: 4285 (1992); Presta et al, J. Immunol., 151: 2623 (1993), the contents of which are incorporated herein by reference in its entirety. In some embodiments, the framework regions of the heavy chain variable region, such as all four framework regions, are derived from the VH4_4-59 germline sequence. In one embodiment, the framework regions may comprise, for example, one, two, three, four or five modifications to the amino acid of the corresponding murine sequence, such as substitutions, such as conservative substitutions. In one embodiment, the framework regions of the light chain variable region, such as all four framework regions, are derived from the VK3_1.25 germline sequence. In one embodiment, the framework regions may comprise, for example, one, two, three, four or five modifications to the amino acid of the corresponding murine sequence, such as substitutions, such as conservative substitutions.

In some aspects, a portion of the CAR composition of the invention comprising an antibody fragment is humanized, retaining high affinity for the target antigen and other advantageous biological properties. According to one aspect of the invention, humanized antibodies and antibody fragments are obtained by using a parental and humanized sequence Dimensional models are prepared by analyzing parental sequences and various conceptual humanized products. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. A computer program that illustrates and exhibits a possible three-dimensional configuration of the candidate immunoglobulin sequences can be utilized. Detection of such expression allows analysis of the possible role of the residue in the function of the candidate immunoglobulin sequence, such as the analysis of residues that affect the ability of the candidate immunoglobulin to bind to the target antigen. In this manner, FR residues can be selected and combined from the accept and input sequences to achieve desired antibody or antibody fragment characteristics, such as increased affinity for the target antigen. In general, CDR residues are directly and most substantially involved in affecting antigen binding.

A humanized antibody or antibody fragment can retain antigenic specificity similar to the original antibody, such as the ability to bind human BCMA in the present invention. In some embodiments, a humanized antibody or antibody fragment can have increased affinity and/or binding specificity for human BCMA.

In one embodiment, the humanized anti-BCMA binding domain of CAR comprises one or more (eg, all three) light chain complementarity determining regions 1 (LC CDR1) of the humanized anti-BCMA binding domain described herein. , light chain complementarity determining region 2 (LC CDR2) and light chain complementarity determining region 3 (LC CDR3), and/or one or more (eg, all three) of the humanized anti-BCMA binding domain described herein Chain complementarity determining region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2) and heavy chain complementarity determining region 3 (HC CDR3), for example comprising one or more, for example all three LC CDRs and one or more For example, the humanized anti-BCMA binding domain of all three HC CDRs. In one embodiment, the humanized anti-BCMA binding domain comprises one or more (eg, all three) heavy chain complementarity determining regions 1 (HC CDR1), heavy, of the humanized anti-BCMA binding domain described herein. The strand complementarity determining region 2 (HC CDR2) and the heavy chain complementarity determining region 3 (HC CDR3), eg, the humanized anti-BCMA binding domain has two variable heavy chain regions, each variable heavy region comprising HC CDR1, HC CDR2 and HC CDR3. In one embodiment, the humanized anti-BCMA binding domain comprises a humanized light chain variable region as described herein (eg, SEQ ID NO: 255 or 257) and/or A human heavy chain variable region (e.g., SEQ ID NO: 255 or 257).

In one aspect, the anti-BCMA binding domain is characterized by a particular functional characteristic or characteristic of the antibody or antibody fragment. For example, in one aspect, a portion of a CAR composition of the invention comprising an antigen binding domain specifically binds to human BCMA.

In one aspect, the antigen binding domain has the same or similar binding specificity to human BCMA as mouse BCMA. In one aspect, the invention relates to an antigen binding domain comprising an antibody or antibody fragment, wherein the antibody binding domain specifically binds to a BCMA protein or a fragment thereof, wherein the antibody or antibody fragment comprises SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148 or a variable light chain and/or a variable heavy chain of the amino acid sequence of SEQ ID NO: 149. In one aspect, the antigen binding domain comprises a mutation selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO :53, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136 SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143. The amino acid sequence of scFv of SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148 or SEQ ID NO: 149. In some aspects, the scFv is adjacent to the leader sequence and is in the same reading frame. In one aspect, the leader sequence is the polypeptide sequence provided as SEQ ID NO: 1.

In one aspect, the anti-BCMA binding domain is a fragment, such as a single chain variable fragment (scFv). In one aspect, the anti-BCMA binding domain is an Fv, Fab, (Fab') 2 or bifunctional (e.g., bispecific) hybrid antibody (e.g., Lanzavecchia et al, Eur. J. Immunol. 17, 105 (1987)) . In one aspect, the antibodies and fragments thereof of the invention bind to the BCMA protein in either wild-type or enhanced affinity.

In some cases, the scFv can be according to methods known in the art (see, for example, Bird et al, (1988) Science 242: 423-426 and Huston et al, (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883) Preparation. The ScFv molecule can be produced by joining together the VH and VL regions using a flexible polypeptide linker. The scFv molecule comprises a linker (eg, a Ser-Gly linker) having an optimized length and/or an amino acid composition. The length of the linker can greatly affect the manner in which the variable regions of the scFv fold and interact. In fact, if a short polypeptide linker (e.g., 5-10 amino acids) is employed, intra-chain folding is prevented. Inter-strand folding is also required to allow the two variable regions to together form a functional epitope binding site. For examples of linker orientation and size, see, for example, Hollinger et al. 1993 Proc Natl Acad. Sci. USA 90:6444-6448; U.S. Patent Application Publication No. 2005/0100543, No. 2005/0175606, No. 2007/0014794 And PCT Publication Nos. WO2006/020258 and WO2007/024715, herein incorporated by reference.

The scFv may include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 between its VL and VH regions. A linker of 20, 25, 30, 35, 40, 45, 50 or more amino acid residues. The linker sequence can comprise any naturally occurring amino acid. In some embodiments, the linker sequence comprises amino acid glycine and serine. In another embodiment, the linker sequence comprises a glycine and a serine repeat unit group, such as (Gly ₄ Ser) _n , wherein n is a positive integer equal to or greater than 1 (SEQ ID NO: 25). In one embodiment, the linker can be (Gly ₄ Ser) ₄ (SEQ ID NO: 27) or (Gly ₄ Ser) ₃ (SEQ ID NO: 28). Changes in the length of the linker can retain or enhance activity and produce superior efficacy in activity studies.

Exemplary human BCMA CAR constructs and antigen binding domains

Exemplary BCMA CAR constructs disclosed herein comprise an scFv (e.g., an scFv as disclosed in Tables 1 or 16), optionally preceded by a leader sequence (e.g., an exemplary leader amino acid and nucleotide sequence, respectively) Is SEQ ID NO: 1 and SEQ ID NO: 12). The sequence of the scFv fragment (SEQ ID NO: 39-53, 129-149 or 263-266, excluding the presence of the leader sequence as appropriate) is provided in Tables 1 or 16 herein. The BCMA CAR construct may further comprise a hinge domain as appropriate, such as a CD8 hinge domain (eg, comprising the amino acid sequence of SEQ ID NO: 2 or encoded by the nucleic acid sequence of SEQ ID NO: 13); a transmembrane structure a domain, such as a CD8 transmembrane domain (eg, comprising the amino acid sequence of SEQ ID NO: 6 or encoded by the nucleotide sequence of SEQ ID NO: 17); an intracellular domain, eg, a 4-1BB intracellular domain (for example comprising the amino acid sequence of SEQ ID NO: 7 or encoded by the nucleotide sequence of SEQ ID NO: 18); and a functional signaling domain, such as a CD3ξ domain (for example comprising SEQ ID NO: 9 or The amino acid sequence of 10, or encoded by the nucleotide sequence of SEQ ID NO: 20 or 21. In certain embodiments, the domains are adjacent and in the same reading frame to form a single fusion protein. In other embodiments, the domains are in separate polypeptides, such as RCAR molecules as described herein.

In certain embodiments, the full length BCMA CAR molecule comprises BCMA-1, BCMA-2, BCMA-3, BCMA-4, BCMA-5, BCMA-6, BCMA-7, BCMA- provided in Table 1 or 16. 8. BCMA-9, BCMA-10, BCMA-11, BCMA-12, BCMA-13, BCMA-14, BCMA-15, 149362, 149363, 149364, 149365, 149366, 149367, 149368, 149369, BCMA_EBB-C1978- A4, BCMA_EBB-C1978-G1, BCMA_EBB-C1979-C1, BCMA_EBB- C1978-C7, BCMA_EBB-C1978-D10, BCMA_EBB-C1979-C12, BCMA_EBB-C1980-G4, BCMA_EBB-C1980-D2, BCMA_EBB-C1978-A10, BCMA_EBB-C1978-D4, BCMA_EBB-C1980-A2, BCMA_EBB-C1981- Amino acid sequence of C3, BCMA_EBB-C1978-G4, A7D12.2, C11D5.3, C12A3.2 or C13F12.1 or a sequence identical thereto (e.g., 95%-99%) or by BCMA-1, BCMA-2, BCMA-3, BCMA-4, BCMA-5, BCMA-6, BCMA-7, BCMA-8, BCMA-9, BCMA-10, BCMA-11, BCMA-12, BCMA-13, BCMA- 14. BCMA-15, 149362, 149363, 149364, 149365, 149366, 149367, 149368, 149369, BCMA_EBB-C1978-A4, BCMA_EBB-C1978-G1, BCMA_EBB-C1979-C1, BCMA_EBB-C1978-C7, BCMA_EBB-C1978- D10, BCMA_EBB-C1979-C12, BCMA_EBB-C1980-G4, BCMA_EBB-C1980-D2, BCMA_EBB-C1978-A10, BCMA_EBB-C1978-D4, BCMA_EBB-C1980-A2, BCMA_EBB-C1981-C3, BCMA_EBB-C1978-G4, The nucleotide sequence of A7D12.2, C11D5.3, C12A3.2 or C13F12.1 or a sequence encoding substantially identical (e.g., 95%-99%).

In certain embodiments, the BCMA CAR molecule or anti-BCMA antigen binding domain comprises BCMA-1, BCMA-2, BCMA-3, BCMA-4, BCMA-5, BCMA-6 as provided in Table 1 or 16. , BCMA-7, BCMA-8, BCMA-9, BCMA-10, BCMA-11, BCMA-12, BCMA-13, BCMA-14, BCMA-15, 149362, 149363, 149364, 149365, 149366, 149367, 149368 , 149369, BCMA_EBB-C1978-A4, BCMA_EBB-C1978-G1, BCMA_EBB-C1979-C1, BCMA_EBB-C1978-C7, BCMA_EBB-C1978-D10, BCMA_EBB-C1979-C12, BCMA_EBB-C1980-G4, BCMA_EBB-C1980-D2 , BCMA_EBB-C1978-A10, BCMA_EBB-C1978-D4, BCMA_EBB-C1980-A2, BCMA_EBB-C1981-C3, BCMA_EBB-C1978-G4, The scFv amino acid sequence of A7D12.2, C11D5.3, C12A3.2 or C13F12.1 (with or without a leader sequence) or substantially identical to any of the foregoing sequences (eg, 95%-99% identical, or at most 20, A sequence of 15, 10, 8, 6, 5, 4, 3, 2 or 1 amino acid change, such as a substitution (e.g., conservative substitution).

In certain embodiments, the BCMA CAR molecule or anti-BCMA antigen binding domain comprises BCMA-1, BCMA-2, BCMA-3, BCMA-4, BCMA-5, BCMA-6 as provided in Table 1 or 16. , BCMA-7, BCMA-8, BCMA-9, BCMA-10, BCMA-11, BCMA-12, BCMA-13, BCMA-14, BCMA-15, 149362, 149363, 149364, 149365, 149366, 149367, 149368 , 149369, BCMA_EBB-C1978-A4, BCMA_EBB-C1978-G1, BCMA_EBB-C1979-C1, BCMA_EBB-C1978-C7, BCMA_EBB-C1978-D10, BCMA_EBB-C1979-C12, BCMA_EBB-C1980-G4, BCMA_EBB-C1980-D2 Heavy chain of BCMA_EBB-C1978-A10, BCMA_EBB-C1978-D4, BCMA_EBB-C1980-A2, BCMA_EBB-C1981-C3, BCMA_EBB-C1978-G4, A7D12.2, C11D5.3, C12A3.2 or C13F12.1 The variable region and/or the light chain variable region, or substantially identical to any of the foregoing sequences (eg, 95%-99% identical, or at most 20, 15, 10, 8, 6, 5, 4, 3, 2, or 1) Amino acid changes, such as the sequence of a substitution (eg, conservative substitution)).

In certain embodiments, the BCMA CAR molecule or anti-BCMA antigen binding domain comprises one, two or three CDRs (eg, HCDR1, HCDR2 and/or HCDR3) from the heavy chain variable region provided in Table 20. And/or from BCMA-1, BCMA-2, BCMA-3, BCMA-4, BCMA-5, BCMA-6, BCMA-7, BCMA-8, BCMA-9, BCMA-10 provided in Table 21. , BCMA-11, BCMA-12, BCMA-13, BCMA-14, BCMA-15, 149362, 149363, 149364, 149365, 149366, 149367, 149368, 149369, BCMA_EBB-C1978-A4, BCMA_EBB-C1978-G1, BCMA_EBB -C1979-C1, BCMA_EBB-C1978- C7, BCMA_EBB-C1978-D10, BCMA_EBB-C1979-C12, BCMA_EBB-C1980-G4, BCMA_EBB-C1980-D2, BCMA_EBB-C1978-A10, BCMA_EBB-C1978-D4, BCMA_EBB-C1980-A2, BCMA_EBB-C1981-C3, One, two or three CDRs of the light chain variable region of BCMA_EBB-C1978-G4, A7D12.2, C11D5.3, C12A3.2 or C13F12.1 (eg LCDR1, LCDR2 and/or LCDR3); or A preceding sequence is substantially identical (eg, 95%-99% identical, or at most 20, 15, 10, 8, 6, 5, 4, 3, 2 or 1 amino acid changes, such as substitutions (eg, conservative substitutions)) The sequence.

In certain embodiments, the BCMA CAR molecule or anti-BCMA antigen binding domain comprises one, two or three CDRs (eg, HCDR1, HCDR2 and/or HCDR3) from the heavy chain variable region provided in Table 22. And/or from BCMA-1, BCMA-2, BCMA-3, BCMA-4, BCMA-5, BCMA-6, BCMA-7, BCMA-8, BCMA-9, BCMA-10 provided in Table 23. , BCMA-11, BCMA-12, BCMA-13, BCMA-14, BCMA-15, 149362, 149363, 149364, 149365, 149366, 149367, 149368, 149369, BCMA_EBB-C1978-A4, BCMA_EBB-C1978-G1, BCMA_EBB -C1979-C1, BCMA_EBB-C1978-C7, BCMA_EBB-C1978-D10, BCMA_EBB-C1979-C12, BCMA_EBB-C1980-G4, BCMA_EBB-C1980-D2, BCMA_EBB-C1978-A10, BCMA_EBB-C1978-D4, BCMA_EBB-C1980 One, two or three CDRs of the light chain variable region of -A2, BCMA_EBB-C1981-C3, BCMA_EBB-C1978-G4, A7D12.2, C11D5.3, C12A3.2 or C13F12.1 (eg LCDR1, LCDR2) And/or LCDR3); or substantially identical to any of the foregoing sequences (eg, 95%-99% identical, or up to 20, 15, 10, 8, 6, 5, 4, 3, 2 or 1 amino acid changes) For example, the sequence of a substitution (eg, conservative substitution)).

In certain embodiments, the BCMA CAR molecule or anti-BCMA antigen binding domain comprises one, two or three CDRs from the heavy chain variable regions provided in Table 24 (eg HCDR1, HCDR2 and/or HCDR3); and/or from BCMA-1, BCMA-2, BCMA-3, BCMA-4, BCMA-5, BCMA-6, BCMA-7, BCMA-8 provided in Table 25. , BCMA-9, BCMA-10, BCMA-11, BCMA-12, BCMA-13, BCMA-14, BCMA-15, 149362, 149363, 149364, 149365, 149366, 149367, 149368, 149369, BCMA_EBB-C1978-A4 , BCMA_EBB-C1978-G1, BCMA_EBB-C1979-C1, BCMA_EBB-C1978-C7, BCMA_EBB-C1978-D10, BCMA_EBB-C1979-C12, BCMA_EBB-C1980-G4, BCMA_EBB-C1980-D2, BCMA_EBB-C1978-A10, BCMA_EBB One, two or one of the light chain variable regions of -C1978-D4, BCMA_EBB-C1980-A2, BCMA_EBB-C1981-C3, BCMA_EBB-C1978-G4, A7D12.2, C11D5.3, C12A3.2 or C13F12.1 Three CDRs (eg, LCDR1, LCDR2, and/or LCDR3); or substantially identical to any of the foregoing sequences (eg, 95%-99% identical, or at most 20, 15, 10, 8, 6, 5, 4, 3, A sequence of 2 or 1 amino acid change, such as a substitution (eg, conservative substitution)).

The sequences of the human CDR sequences of the scFv domain with respect to the heavy chain variable domain are shown in Tables 20, 22 and 24, and with respect to the light chain variable domains, are shown in Tables 21, 23 and 25. "ID" represents the corresponding SEQ ID NO of each CDR.

In certain embodiments, a CAR molecule (eg, a CAR nucleic acid or CAR polypeptide) or a BCMA binding domain described herein comprises: (1) one, two or three light chain (LC) CDRs selected from one of the following: : (i) LC CDR1 of SEQ ID NO: 504 of BCMA-4 CAR (139103), LC CDR2 of SEQ ID NO: 544 and LC CDR3 of SEQ ID NO: 584; (ii) BCMA-10 CAR (139109) LC CDR1, SEQ of SEQ ID NO:514 ID NO: 554 LC CDR2 and SEQ ID NO: 594 LC CDR3; (iii) BCMA-13 CAR (139112) SEQ ID NO: 516 LC CDR1, SEQ ID NO: 556 LC CDR2 and SEQ ID NO: LC CDR3 of 596; or (iv) LC CDR1 of SEQ ID NO: 518 of BCMA-15 CAR (139114), LC CDR2 of SEQ ID NO: 558, and LC CDR3 of SEQ ID NO: 598; and/or (2) One, two or three heavy chain (HC) CDRs from one of: (i) HC CDR1 of SEQ ID NO: 384 of BCMA-4 CAR (139103), HC CDR2 of SEQ ID NO: 424 and SEQ ID NO: 464 of HC CDR3; (ii) BCMA-10 CAR (139109) SEQ ID NO: 394, HC CDR1, SEQ ID NO: 434, HC CDR2, and SEQ ID NO: 474, HC CDR3; (iii) BCMA- 13 CAR (139112) SEQ ID NO: 396, HC CDR1, SEQ ID NO: 436, HC CDR2, and SEQ ID NO: 476, HC CDR3; or (iv) BCMA-15 (139114), SEQ ID NO: 398 HC CDR1, HC CDR2 of SEQ ID NO: 438 and HC CDR3 of SEQ ID NO:478.

In certain embodiments, a CAR molecule (eg, a CAR nucleic acid or a CAR polypeptide) described herein comprises: (1) one, two or three light chain (LC) CDRs selected from one of: (i) BCMA -4 CAR (139103) SEQ ID NO: 744 LC CDR1, SEQ ID NO: 784 LC CDR2 and SEQ ID NO: 824 LC CDR3; (ii) BCMA-10 CAR (139109) SEQ ID NO: 754 LC CDR1, LC CDR2 of SEQ ID NO: 794 and LC CDR3 of SEQ ID NO: 834; (iii) LC CDR1 of SEQ ID NO: 756 of BCMA-13 CAR (139112), LC CDR2 of SEQ ID NO: 796 And LC CDR3 of SEQ ID NO: 836; or (iv) LC CDR1 of SEQ ID NO: 758 of BCMA-15 CAR (139114), LC CDR2 of SEQ ID NO: 798, and LC CDR3 of SEQ ID NO: 838; / or (2) one, two or three heavy chain (HC) CDRs selected from one of the following: (i) HC CDR1 of SEQ ID NO: 624 of BCMA-4 CAR (139103), HC CDR2 of SEQ ID NO: 664 and HC CDR3 of SEQ ID NO: 704; (ii) SEQ of BCMA-10 CAR (139109) ID NO: 634, HC CDR1, SEQ ID NO: 674, HC CDR2, and SEQ ID NO: 714, HC CDR3; (iii) BCMA-13 CAR (139112), SEQ ID NO: 636, HC CDR1, SEQ ID NO: HC CDR2 of 676 and HC CDR3 of SEQ ID NO: 716; or (iv) HC CDR1 of SEQ ID NO: 638 of BCMA-15 (139114), HC CDR2 of SEQ ID NO: 678 and HC of SEQ ID NO: 718 CDR3.

In certain embodiments, a CAR molecule (eg, a CAR nucleic acid or a CAR polypeptide) described herein comprises: (1) one, two or three light chain (LC) CDRs selected from one of: (i) BCMA -4 CAR (139103) SEQ ID NO: 984 LC CDR1, SEQ ID NO: 1024 LC CDR2 and SEQ ID NO: 1064 LC CDR3; (ii) BCMA-10 CAR (139109) SEQ ID NO: 994 LC CDR1, LC CDR2 of SEQ ID NO: 1034 and LC CDR3 of SEQ ID NO: 1074; (iii) LC CDR1 of SEQ ID NO: 996 of BCMA-13 CAR (139112), LC CDR2 of SEQ ID NO: 1036 And LC CDR3 of SEQ ID NO: 1076; or (iv) LC CDR1 of SEQ ID NO: 998 of BCMA-15 CAR (139114), LC CDR2 of SEQ ID NO: 1038, and LC CDR3 of SEQ ID NO: 1078; / or (2) one, two or three heavy chain (HC) CDRs selected from one of the following: (i) HC CDR1 of SEQ ID NO: 864 of BCMA-4 CAR (139103), SEQ ID NO: 904 HC CDR2 and HC CDR3 of SEQ ID NO:944; (ii) HC CDR1 of SEQ ID NO:874 of BCMA-10 CAR (139109), HC CDR2 of SEQ ID NO:914 and HC CDR3 of SEQ ID NO:954 (iii) HC CDR1 of SEQ ID NO:876 of BCMA-13 CAR (139112), HC CDR2 of SEQ ID NO:916, and HC CDR3 of SEQ ID NO:956; or (iv) HC CDR1 of SEQ ID NO:878 of BCMA-15 (139114), HC CDR2 of SEQ ID NO:918 and HC CDR3 of SEQ ID NO:958.

In an embodiment, an anti-BCMA CAR construct, such as a human or humanized anti-BCMA CAR construct, is produced using methods described herein, for example, as described in Example 4. Exemplary anti-BCMA scFvs include, but are not limited to, BCMA-1, BCMA-2, BCMA-3, BCMA-4, BCMA-5, BCMA-6, BCMA-7, BCMA-8, BCMA-9, BCMA- 10. BCMA-11, BCMA-12, BCMA-13, BCMA-14 and BCMA-15. The sequence of the human anti-BCMA scFv fragment (SEQ ID NOS: 39-52) is provided in Table 1 (and the nomenclature is provided in Table 2).

In an embodiment, a scFv fragment, such as a human scFv fragment (eg, SEQ ID NO: 39-52), is used in combination with additional sequences, such as the sequences shown below, to generate a complete BCMA CAR construct (eg, SEQ ID NO: 99-113) ).

It is to be noted that the invention encompasses scFv fragments as described herein, such as in Tables 1 and 16 or in SEQ ID NOs: 39-53, 129-149, 263-266, 271 or 273, without a leader sequence (eg, without SEQ ID) NO: amino acid sequence of 1 or nucleotide sequence of SEQ ID NO: 12). And s. The amino acid sequence of SEQ ID NO: 1 or the nucleotide sequence of SEQ ID NO: 12).

Leader sequence (amino acid sequence) (SEQ ID NO: 1)

MALPVTALLLPLALLLHAARP

Leader sequence (nucleic acid sequence) (SEQ ID NO: 12)

ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCTCTGGCTCTGCTGCTGCATGCCGCTAGACCC

CD8 hinge (amino acid sequence) (SEQ ID NO: 2)

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

CD8 hinge (nucleic acid sequence) (SEQ ID NO: 13)

CD8 transmembrane (amino acid sequence) (SEQ ID NO: 6)

IYIWAPLAGTCGVLLLSLVITLYC

CD8 transmembrane (nucleic acid sequence) (SEQ ID NO: 17)

ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC

4-1BB intracellular domain (amino acid sequence) (SEQ ID NO: 7)

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

4-1BB intracellular domain (nucleic acid sequence) (SEQ ID NO: 18)

CD28 intracellular domain (amino acid sequence) (SEQ ID NO: 1104)

RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 1104)

CD28 intracellular domain (nucleotide sequence) (SEQ ID NO: 1105) (SEQ ID NO: 1105)

ICOS intracellular domain (amino acid sequence) (SEQ ID NO: 1106)

T K K K Y S S S H H D P N G E Y M F M R A V N T A K K S R L T D V T L (SEQ ID NO: 1106)

ICOS intracellular domain (nucleotide sequence) (SEQ ID NO: 1107)

ACAAAAAAGAAGTATTCATCCAGTGTGCACGACCCTAACGGTGAATACATGTTCATGAGAGCAGTGAACACAGCCAAAAAATCCAGACTCACAGATGTGACCCTA (SEQ ID NO:1107)

CD3ζ domain (amino acid sequence) (SEQ ID NO: 9)

RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

CD3ζ (nucleic acid sequence) (SEQ ID NO: 20)

CD3ζ domain (amino acid sequence; NCBI reference sequence NM_000734.3) (SEQ ID NO: 10)

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

CD3ζ (nucleic acid sequence; NCBI reference sequence NM_000734.3); (SEQ ID NO: 21)

IgG4 hinge (amino acid sequence) (SEQ ID NO: 36)

IgG4 hinge (nucleotide sequence) (SEQ ID NO: 37)

In the Examples, a CAR scFv fragment was cloned into a lentiviral vector to generate a full-length CAR construct for expression (SEQ ID NO: 11) in a single coding frame and using a promoter, such as the EF1α promoter.

EF1α promoter (SEQ ID NO: 11).

Gly/Ser (SEQ ID NO: 25)

GGGGS

Gly/Ser (SEQ ID NO: 26): This sequence can cover 1-6 "Gly Gly Gly Gly Ser" repeat units

GGGGSGGGGS GGGGSGGGGS GGGGSGGGGS

Gly/Ser (SEQ ID NO: 27)

GGGGSGGGGS GGGGSGGGGS

Gly/Ser (SEQ ID NO: 28)

GGGGSGGGGS GGGGS

Gly/Ser (SEQ ID NO: 29)

GGGS

Poly A: (A) ₅₀₀₀ (SEQ ID NO: 30)

Poly A: (T) ₁₀₀ (SEQ ID NO: 31)

Poly A: (T) ₅₀₀₀ (SEQ ID NO: 32)

Poly A: (A) ₅₀₀₀ (SEQ ID NO: 33)

Poly A: (A) ₄₀₀ (SEQ ID NO: 34)

Poly A: (A) ₂₀₀₀ (SEQ ID NO: 35)

Gly/Ser (SEQ ID NO: 38): This sequence can cover 1-10 "Gly Gly Gly Ser" repeat units

GGGSGGGSGG GSGGGSGGGS GGGSGGGSGG GSGGGSGGGS

The exemplary BCMA scFv domains and the amino acid and nucleic acid sequences of exemplary BCMA CAR molecules are provided in Table 1.

Table 2 below indicates the nickname of the BCMA CAR construct relative to the number of DNA IDs also listed in Table 1.

In an embodiment, the VH and VL sequences from PCT Publication No. WO 2012/0163805, the contents of which are hereby incorporated by reference in its entirety, are used, for example, as described in Examples 2 and 3 As a result of pBCMA3 and pBCMA4 CAR, other exemplary BCMA CAR constructs were generated. A schematic of one of the exemplary BCMA constructs (BCMA-3NP and BCMA-4NP) is shown in Figure 10A. The orientations of the VH and VL chains of the two constructs are different (Fig. 10B). Exemplary BCMA CAR constructs and their corresponding DNA IDs are shown in Table 3 below.

In the examples, other exemplary BCMA CAR constructs can also be generated using the VH and VL sequences found in Table 16. Exemplary scFv domains and linker sequences comprising the VH and VL domains and the amino acid sequence of the full length CAR are also found in Table 16.

In an embodiment, the nucleic acid sequence of an exemplary humanized anti-BCMA scFv wherein VH is before VL (H2L, eg, pBCMA 2 and pBCMA 4) is as follows: (SEQ ID NO: 272)

The corresponding amino acid sequence of an exemplary humanized anti-BCMA scFv of VH before VL (H2L, such as pBCMA 2 and pBCMA 4) is as follows: (SEQ ID NO: 271)

In an embodiment, the nucleic acid sequence of an exemplary humanized anti-BCMA scFv wherein VL is before VH (L2H, eg, pBCMA1 and pBCMA3) is as follows: (SEQ ID NO: 274)

The corresponding amino acid sequence of an exemplary humanized anti-BCMA scFv in which VL is before VH (L2H, such as pBCMA1 and pBCMA3) is as follows: (SEQ ID NO: 273)

The CAR scFv fragment was optionally cloned into a lentiviral vector to form a full-length CAR construct in a single coding frame and using the EF1 alpha promoter for expression (SEQ ID NO: 11).

The CAR construct may comprise a Gly/Ser linker having one or more of the following sequences: GGGGS (SEQ ID NO: 25); covering 1-6 "Gly Gly Gly Gly Ser" repeat units, such as GGGGSGGGGS GGGGSGGGGS GGGGSGGGGS ( SEQ ID NO: 26); GGGGSGGGGS GGGGSGGGGS (SEQ ID NO: 27); GGGGSGGGGS GGGGS (SEQ ID NO: 28); GGGS (SEQ ID NO: 29); or encompasses 1-10 "Gly Gly Gly Ser" repeat units For example, GGGSGGGSGG GSGGGSGGGS GGGSGGGSGG GSGGGSGGGS (SEQ ID NO: 38).

In an embodiment, the CAR construct comprises a poly A sequence, eg, a sequence encompassing 50-5000 or 100-5000 adenines (eg, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 34, or SEQ ID NO: 35) or a sequence encompassing 50-5000 thymines (eg, SEQ ID NO: 31, SEQ ID NO: 32). Alternatively, the CAR construct can include, for example, a linker comprising the sequence GSTSGSGKPGSGEGSTKG (SEQ ID NO: 1108).

Bispecific CAR

In one embodiment, the multispecific antibody molecule is a bispecific antibody molecule. Bispecific antibodies have specificity for no more than two antigens. The bispecific antibody molecule is characterized in that the first immunoglobulin variable domain sequence has binding specificity for the first epitope and the second immunoglobulin variable domain sequence has binding specificity for the second epitope Sex. In one embodiment, the first and second epitopes are on the same antigen, such as the same protein (or a subunit of a polyprotein). In one embodiment, the first and second epitopes overlap. In one embodiment, the first and second epitopes do not overlap. In one embodiment, the first and second epitopes are on different antigens, such as different proteins (or different subunits of the polyprotein). In one embodiment, the bispecific antibody molecule comprises a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity for the first epitope and binding specificity for the second epitope Heavy chain variable domain sequences and light chain variable domain sequences. In one embodiment, the bispecific antibody molecule comprises a half antibody having binding specificity for the first epitope and a half antibody having binding specificity for the second epitope. In one embodiment, the bispecific antibody molecule comprises a half antibody or fragment thereof having binding specificity for a first epitope and a half antibody or fragment thereof having binding specificity for a second epitope. In one embodiment, the bispecific antibody molecule comprises an scFv having a binding specificity for a first epitope or a fragment thereof and an scFv having a binding specificity for a second epitope or a fragment thereof.

In certain embodiments, the antibody molecule is multispecific (eg, bispecific or trispecific) Sex) antibody molecule. Schemes for the production of bispecific or heterodimeric antibody molecules are known in the art; including, but not limited to, the "杵臼" method as described, for example, in US Pat. No. 5,731,168; such as, for example, WO 09/089004, WO Electrostatically directed Fc pairing as described in 06/106905 and WO 2010/129304; as described, for example, in the stock exchange engineering domain (SEED) heterodimer described in WO 07/110205; as for example WO 08/119353, WO Fab arm exchange as described in 2011/131746 and WO 2013/060867; dual antibody conjugates, for example by antibody cross-linking using heterobifunctional reagents having an amine-reactive group and a sulfhydryl-reactive group, A bispecific structure, as described, for example, in US Pat. No. 4,433,059; the production of a half antibody (heavy-light chain pair or Fab) from different antibodies by reductive and oxidative cycling of disulfide bonds between two heavy chains Bispecific antibody determinants, as described, for example, in U.S. Patent 4,444,878; trifunctional antibodies, e.g., three Fab' fragments, are cross-linked via a sulfhydryl reactive group, as described, for example, in U.S. Patent 5,237,743; biosynthetic binding proteins, for example via C-terminal tail, preferably via disulfide or amine Sexually chemically cross-linking cross-linked scFv pairs, as described, for example, in US Pat. No. 5,534, 254; bifunctional antibodies, such as different combinations of substituted constant domains dimerized via leucine zippers (eg, c-fos and c-jun) Specific Fab fragments, as described, for example, in US Pat. No. 5,582,996; bispecific and oligospecific monovalent and oligoreceptors, for example, via a polypeptide between the CH1 region of one antibody and the VH region of another antibody normally associated with the light chain The VH-CH1 region of two antibodies (two Fab fragments) linked by a spacer, as described, for example, in US 5,591,828; the bispecific DNA-antibody conjugate, for example, cross-linking via antibodies or Fab fragments of a double-stranded DNA , as described, for example, in US Pat. No. 5,365,602; bispecific fusion proteins, for example, expression constructs comprising two scFvs having a hydrophilic helical peptide linker and having a complete constant region, as described, for example, in US5637481; multivalent and multispecific A sex-binding protein, such as a polypeptide dimer having a first domain comprising a binding region of an Ig heavy chain variable region and a second domain comprising a binding region of an Ig light chain variable region, generally referred to as a bifunctional antibody ( Also covers double Of higher order structure, trispecific or tetra-specific molecules of), for example as described in US5837242; minibody construct, which is connected to VL And the VH chain, the VL and VH chains are further linked to the antibody hinge region and the CH3 region with a peptide spacer, which can be dimerized to form a bispecific/multivalent molecule, as described, for example, in US Pat. No. 5,381,821; and a short peptide linker (eg, 5 or 10 amino acids) linked or non-linker VH and VL domains in either orientation, which can form dimers to form bispecific bifunctional antibodies; trimers and tetramers such as eg Further described in US Pat. No. 5,844,094; a VH domain (or a VL domain in a family member) linked by a peptide bond having a crosslinkable group at the C-terminus to further associate with the VL domain to form a series of FVs (or scFvs) ), as described, for example, in US5864019; and single-stranded binding polypeptides in which the VH and VL domains are linked via a peptide linker are combined into a multivalent structure via non-covalent or chemical cross-linking to form, for example, using scFV or a bifunctional antibody type format. The same bivalent, heterobivalent, trivalent, and tetravalent structures are as described, for example, in U.S. Patent 5,966,620. Other exemplary multi-specificity and bispecific molecules and methods for their preparation are found in, for example, US Pat. No. 5,910, 573, US Pat. No. 5, 932, 448, US Pat. No. 5, 591, 908, US Pat. No. 5, 989, 830, US Pat. No. 6,005, 079, US Pat. No. 6, 239, 259, US Pat. No. 6, 293, 435, US Pat. No. 6,633,396, US Pat. No. 6,472, 198, US Pat. No. 6, 651, 663, US Pat. No. 6,674, s, US Pat. US7527787, US7534866, US7612181, US2002004587A1, US2002076406A1, US2002103345A1, US2003207346A1, US2003211078A1, US2004219643A1, US2004220388A1, US2004242847A1, US2005003403A1, US2005004352A1, US2005069552A1, US2005079170A1, US2005100543A1, US2005136049A1, US2005136051A1, US2005163782A1, US2005266425A1, US2006083747A1, US2006120960A1, US2006204493A1, US2006263367A1, US2007004909A1 US2007087381A1, US2007128150A1, US2007141049A1, US2007154901A1, US2007274985A1, US2008050370A1, US2008069820A1, US2008152645A1, US2008171855A1, US2008241884A1, US2008254512A1 US2008260738A1, US2009130106A1, US2009148905A1, US2009155275A1, US2009162359A1, US2009162360A1, US2009175851A1, US2009175867A1, US2009232811A1, US2009234105A1, US2009263392A1, US2009274649A1, EP346087A2, WO0006605A2, WO02072635A2, WO04081051A1, WO06020258A2, WO2007044887A2, WO2007095338A2, WO2007137760A2, WO2008119353A1, WO2009021754A2, WO2009068630A1, WO9103493A1, WO9323537A1 WO9409131A1, WO9412625A2, WO9509917A1, WO9637621A2, WO9964460A1. The content of the above-referenced application is hereby incorporated by reference in its entirety.

Within each antibody or antibody fragment (eg, scFv) of a bispecific antibody molecule, VH can be upstream or downstream of VL. In some embodiments, upstream of the antibody or antibody fragment (e.g., scFv) are configured such that VH (VH ₁₎ is configured such that VL (VL in which VL (VL ₁₎ upstream and downstream of the antibody or antibody fragment (e.g., scFv) ₂ ) upstream of its VH (VH ₂ ), such that the entire bispecific antibody molecule has the configuration VH ₁ -VL ₁ -VL ₂ -VH ₂ . In other embodiments, the upstream antibodies or antibody fragments (e.g. scFv) are configured such that VL (VL ₁₎ in the (VH ₁₎ upstream of which the VH, and downstream of antibodies or antibody fragments (e.g. scFv) are configured such that VH (VH ₂ ) upstream of its VL (VL ₂ ), such that the entire bispecific antibody molecule has the configuration VL ₁ -VH ₁ -VH ₂ -VL ₂ . Optionally, if the construct is configured as VH ₁ -VL ₁ -VL ₂ -VH ₂ , the linker is placed between two antibodies or antibody fragments (eg, scFv), such as between VL ₁ and VL ₂ , or if constructed The body configuration is VL ₁ -VH ₁ -VH ₂ -VL ₂ and is placed between VH ₁ and VH ₂ . The linker can be a linker as described herein, such as a (Gly ₄ -Ser) n linker, wherein n is 1, 2, 3, 4, 5 or 6, preferably 4 (SEQ ID NO: 26). In general, the linker between the two scFvs should be long enough to avoid mismatching between the domains of the two scFvs. Optionally, the linker is placed between the VL and VH of the first scFv. Optionally, the linker is placed between the VL and VH of the second scFv. In a construct having a plurality of linkers, any two or more linkers may be the same or different. Thus, in some embodiments, the bispecific CAR comprises VL, VH and optionally one or more linkers, the configuration being as described herein.

In one aspect, the bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence, such as an scFv, which has binding specificity for BCMA, eg, as described herein, for example as in Table 1 or Table 16. An scFv as described, or comprising a light chain CDR and/or heavy chain CDR from a BCMA scFv as described herein; and a second immunoglobulin variable domain sequence having a second epitope on a different antigen Binding specificity. In some aspects, the second immunoglobulin variable domain sequence has binding specificity for an antigen expressed on AML cells, such as an antigen other than BCMA. For example, the second immunoglobulin variable domain sequence has binding specificity for CD123. As another example, the second immunoglobulin variable domain sequence has binding specificity for CLL-1. As another example, the second immunoglobulin variable domain sequence has binding specificity for CD34. As another example, the second immunoglobulin variable domain sequence has binding specificity for FLT3. For example, the second immunoglobulin variable domain sequence has binding specificity for the folate receptor beta. In some aspects, the second immunoglobulin variable domain sequence has an antigen on a B cell, such as CD10, CD19, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b or CD79a. Binding specificity.

Chimeric TCR

In one aspect, the anti-BCMA antibodies and antibody fragments of the invention (such as those disclosed in Tables 1 and 16) can be grafted to a T cell receptor ("TCR") chain, such as one or more of a TCR alpha or TCR beta chain. A constant domain to produce a chimeric TCR that specifically binds to BCMA. Without being bound by theory, the Schindler chimeric TCR will signal via the TCR complex upon antigen binding. For example, a BCMA scFv as disclosed herein can be grafted to a TCR chain, such as a constant domain of a TCR alpha chain and/or a TCR beta chain (eg, at least a portion of an extracellular constant domain), a transmembrane domain, and a cytoplasmic domain. As another example, BCMA antibody tablets A segment, such as a VL domain as described herein, can be grafted to a constant domain of a TCR alpha chain, and a BCMA antibody fragment, such as a VH domain as described herein, can be grafted to a constant domain (or VL structure) of a TCR beta chain. The domain can be grafted to the constant domain of the TCR beta chain and the VH domain can be grafted to the TCR alpha chain). As another example, the CDRs of an anti-BCMA antibody or antibody fragment, for example, the CDRs of an anti-BCMA antibody or antibody fragment as described in Table 20, 21, 22, 23, 24 or 25 can be grafted to TCR alpha and/or beta The strands are produced to produce a chimeric TCR that specifically binds to BCMA. For example, an LCDR disclosed herein can be grafted to a variable domain of a TCR alpha chain and the HCDRs disclosed herein can be grafted to a variable domain of a TCR beta chain, or vice versa. Such chimeric TCRs can be by methods known in the art (e.g., Willemsen RA et al, Gene Therapy 2000; 7: 1369-1377; Zhang T et al, Cancer Gene Ther 2004; 11: 487-496; Aggen Et al., Gene Ther. 2012 Apr; 19(4): 365-74).

Transmembrane domain

In various embodiments, with respect to a transmembrane domain, a CAR can be designed to comprise a transmembrane domain linked to the extracellular domain of a CAR. The transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, such as one or more amino acids associated with the extracellular region of the protein from which the transmembrane is derived (eg, 1, 2) , 3, 4, 5, 6, 7, 8, 9, 10 up to 15 extracellular regions of amino acids) and/or one or more additional associated with the intracellular region of the protein from which the transmembrane protein is derived Amino acids (eg 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 intracellular amino acids). In one aspect, the transmembrane domain is a domain associated with one of the other domains of the CAR used. In some cases, the transmembrane domain may be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domain of the same or different surface membrane proteins, eg, to make the complex with the receptor complex The interaction of members is minimized. In one aspect, the transmembrane domain is capable of dimerizing with another CAR on the surface of a cell that exhibits CAR, such as a CART cell. In various aspects, the amino acid sequence of the transmembrane domain may be modified or substituted to allow for the presence of cells in the same CAR, such as those present in CART. The interaction of the binding domains of the bioconjugates is minimized.

Transmembrane domains can be derived from natural or recombinant sources. If the source is native, the domain can be derived from any membrane-bound protein or transmembrane protein. In one aspect, the transmembrane domain is capable of signaling to the intracellular domain as long as the CAR binds to the target. The transmembrane domain specifically for use in the present invention may comprise, for example, at least a transmembrane region: alpha, beta or ζ chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8 (eg, CD8 alpha, CD8 beta) , CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. In some embodiments, the transmembrane domain can comprise a transmembrane region of at least, for example, a co-stimulatory molecule: MHC class I molecule, TNF receptor protein, immunoglobulin-like protein, cytokine receptor, integrin, signaling Lymphocyte activating molecule (SLAM protein), activated NK cell receptor, BTLA, Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a/ CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46 , CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1 , ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile) ), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1 CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a and CD83-specific binding of the ligand.

In some cases, a transmembrane domain can be linked to the extracellular region of a CAR, such as the antigen binding domain of a CAR, via a hinge (eg, a hinge from a human protein). For example, in one embodiment, the hinge can be a human Ig (immunoglobulin) hinge, such as an IgG4 hinge, or a CD8a hinge. In one embodiment, the hinge or spacer comprises (eg, consists of) the amino acid sequence of SEQ ID NO: 2. In one aspect, the transmembrane domain comprises (eg, consists of) the transmembrane domain of SEQ ID NO: 6.

In one aspect, the hinge or spacer comprises an IgG4 hinge. For example, in one embodiment, the hinge or spacer comprises a hinge of the following amino acid sequence: (SEQ ID NO: 3). In some embodiments, the hinge or spacer comprises a hinge encoded by the following nucleotide sequence: (SEQ ID NO: 14).

In one aspect, the hinge or spacer contains an IgD hinge. For example, in one embodiment, the hinge or spacer comprises a hinge of the following amino acid sequence: (SEQ ID NO: 4). In some embodiments, the hinge or spacer comprises a hinge encoded by the following nucleotide sequence: (SEQ ID NO: 15).

In one aspect, the transmembrane domain can be a recombinant transmembrane domain, in which case it will primarily comprise hydrophobic residues such as leucine and proline. In one aspect, a triad of phenylalanine, tryptophan, and valine can be found at each end of the recombinant transmembrane domain.

Depending on the case, a short oligopeptide or polypeptide linker of 2 to 10 amino acids in length can form a link between the transmembrane domain of the CAR and the cytoplasmic domain. The glycine-serine dimer provides a particularly suitable linker. For example, in one aspect, the linker comprises the amino acid sequence GGGGSGGGGS (SEQ ID NO: 5). In some embodiments, the linker is encoded by the nucleotide sequence GGTGGCGGAGGTTCTGGAGGTGGAGGTTCC (SEQ ID NO: 16).

In one aspect, the hinge or spacer contains a KIR2DS2 hinge.

Cytoplasmic domain

The cytoplasmic or cytoplasmic domain of the CAR of the invention comprises an intracellular signaling domain. The intracellular signaling domain is generally responsible for the activation of at least one of the normal effector functions of immune cells that have been introduced into the CAR.

Examples of intracellular signaling domains for use in the CAR of the invention include cytoplasmic sequences that cooperate to initiate signal transduction of T cell receptors (TCRs) and co-receptors after antigen receptor engagement, and such Any derivative or variant of the sequence and the ability to have the same functionality Any recombinant sequence.

It is known that signals generated by separate TCRs are not sufficient to fully activate T cells and also require secondary and/or costimulatory signals. Thus, T cell activation can be referred to as being mediated by two distinct classes of cytoplasmic signaling sequences: initiation of antigen-dependent primary activators via TCR (primary intracellular signaling domain) and functioning in an antigen-independent manner to provide Secondary or co-stimulators (secondary cytoplasmic domains, such as costimulatory domains).

The primary signaling domain regulates the primary activation of the TCR complex in a stimulatory or inhibitory manner. The major intracellular signaling domain that functions in a stimulatory manner may contain a signaling motif called an immunoreceptor tyrosine-based activation motif or ITAM.

Examples of ITAMs containing a major intracellular signaling domain that are particularly suitable for use in the present invention include: TCR, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS") ), FcεRI, DAP10, DAP12 and CD66d. In one embodiment, the CAR of the invention comprises an intracellular signaling domain, such as the major signaling domain of CD3-ζ.

In one embodiment, the primary signaling domain comprises a modified ITAM domain, such as a mutant ITAM domain that has altered (eg, increased or decreased) activity compared to a native ITAM domain. In one embodiment, the primary signaling domain comprises a major intracellular signaling domain comprising a modified ITAM, such as a major intracellular signaling domain that contains an optimized and/or truncated ITAM. In an embodiment, the primary signaling domain comprises one, two, three, four or more ITAM primitives.

Other examples of molecules containing a major intracellular signaling domain that are particularly suitable for use in the present invention include those of DAP10, DAP12, and CD32.

The intracellular signaling domain of CAR may comprise a major signaling domain, such as the CD3-ζ signaling domain itself or it may be combined with any other desired intracellular signaling domain in the context of a CAR suitable for use in the present invention. For example, the intracellular signaling domain of CAR can comprise a major signaling domain, such as a CD3 ζ chain moiety, and a common thorn Excitation signal transduction domain. A costimulatory signaling domain refers to a portion of a CAR that contains the intracellular domain of a costimulatory molecule. The costimulatory molecule is a cell surface molecule other than the antigen receptor or its ligand required for the lymphocyte to efficiently react with the antigen. Examples of such molecules include MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activating molecules (SLAM proteins), activated NK cell receptors, BTLA, Toll Ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3, CDS, ICAM- 1. ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1 , CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA -1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1 CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SL P-76, PAG/Cbp, CD19a and ligands that specifically bind to CD83 and analogs thereof. For example, CD27 co-stimulation has demonstrated that in vitro enhances the expansion, effector function, and survival of human CART cells and enhances human T cell persistence and antitumor activity in vivo (Song et al. Blood. 2012; 119(3) :696-706). The intracellular signaling sequences within the cytoplasmic portion of the CAR of the invention can be linked to each other in a random or specified sequence. Depending on the case, short oligopeptides or polypeptide linkers of 2 to 10 amino acids in length (eg 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids) can be signalled intracellularly A connection is formed between the sequences. In one embodiment, A glycine-serine dimer can be used as a suitable linker. In one embodiment, a single amino acid such as alanine or glycine can be used as a suitable linker.

In one aspect, the intracellular signaling domain is designed to comprise two or more, eg, 2, 3, 4, 5 or more co-stimulatory signaling domains. In one embodiment, two or more (eg, 2, 3, 4, 5 or more) costimulatory signaling domains are separated by a linker molecule (eg, a linker molecule as described herein). In one embodiment, the intracellular signaling domain comprises two costimulatory signaling domains. In some embodiments, the linker molecule is a glycine residue. In some embodiments, the linker is an alanine residue.

In one aspect, the intracellular signaling domain is designed to comprise a CD3-ζ signaling domain and a CD28 signaling domain. In one aspect, the intracellular signaling domain is designed to comprise a CD3-ζ signaling domain and a 4-1BB signaling domain. In one aspect, the signaling domain of 4-1BB is the signaling domain of SEQ ID NO:7. In one aspect, the signaling domain of CD3-ζ is the signaling domain of SEQ ID NO: 9 (mutant CD3ζ) or SEQ ID NO: 10 (wild-type human CD3ζ).

In one aspect, the intracellular signaling domain is designed to comprise a CD3-ζ signaling domain and a CD27 signaling domain. In one aspect, the signaling domain of CD27 comprises the amino acid sequence QRRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEPACSP (SEQ ID NO: 8). In one aspect, the signaling domain of CD27 consists of The nucleic acid sequence of (SEQ ID NO: 19) is encoded.

In one aspect, the intracellular signaling domain is designed to contain a CD3-ξ signal The conduction domain and the signaling domain of CD28. In one aspect, the signaling domain of CD28 comprises the amino acid sequence of SEQ ID NO: 1104. In one aspect, the signaling domain of CD28 is encoded by the nucleic acid sequence of SEQ ID NO: 1105.

In one aspect, the intracellular signaling domain is designed to comprise the signaling domain of CD3-ξ and the signaling domain of ICOS. In one aspect, the signaling domain of ICOS comprises the amino acid sequence of SEQ ID NO: 1106. In one aspect, the signaling domain of ICOS is encoded by the nucleic acid sequence of SEQ ID NO: 1107.

In one aspect, the CAR-expressing cells described herein can further comprise a second CAR, for example including, for example, against the same target (BCMA) or a different target (eg, CD19, CD20 or CS-1, or other multiple bone marrow) The second target antigen binding domain of a tumor target, such as kappa light chain, CD138, Lewis Y antigen or CD38 (Garfall et al, Discovery Medicine, 2014, 17 (91): 37-46) CAR. In one embodiment, the cell expressing CAR comprises a first CAR that targets the first antigen and includes an intracellular signaling domain having a costimulatory signaling domain other than the primary signaling domain, and targeting the second different Antigen and includes a second CAR having a major signaling domain rather than an intracellular signaling domain of a costimulatory signaling domain. While not wishing to be bound by theory, a co-stimulatory signaling domain (eg, 4-1BB, CD28, CD27ICOS, or OX-40) is placed on the first CAR and a major signaling domain (eg, CD3ζ) is placed in the second CAR. The CAR activity can be limited to cells that express two targets. In one embodiment, the CAR-expressing cell comprises a first BCMA CAR comprising a BCMA binding domain, a transmembrane domain, and a costimulatory domain; and a second CAR that targets an antigen other than BCMA (eg, in leukemia) Or antigens expressed on lymphoma cells, such as CD19, CD20, CS-1, kappa light chain, CD139, Lewis Y antigen or CD38) and include antigen binding domains, transmembrane domains, and major signaling domains. In another embodiment, the CAR-expressing cell comprises a first BCMA CAR comprising a BCMA binding domain, a transmembrane domain and a major signaling domain; and a second CAR, which is targeted to besides BCMA An antigen (eg, an antigen expressed on leukemia or lymphoma cells, eg, CD19, CD20, CS-1, kappa light chain, CD139, Lewis Y antigen, or CD38) and includes an antigen binding domain, transmembrane structure for the antigen Domain and co-stimulatory signaling domains. In one embodiment, the cell expressing CAR comprises BCMA CAR as described herein and CAR (CD19 CAR) targeting CD19.

In one embodiment, the cells expressing CAR comprise BCMA CAR and inhibit CAR as described herein. In one embodiment, the inhibitory CAR comprises an antigen binding domain that binds to an antigen found on normal cells (eg, normal cells that also exhibit mesothelin) rather than cancer cells. In one embodiment, the inhibitory CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular domain of an inhibitory molecule. For example, the intracellular domain that inhibits CAR can be PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (eg, CEACAM-1, CEACAM-3, and/or CEACAM-5), LAG3, VISTA, BTLA , TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine And the intracellular domain of TGFR β.

In one embodiment, when a cell expressing CAR comprises two or more different CARs, the antigen binding domains of different CARs may cause the antigen binding domains not to interact with each other. For example, a cell expressing the first and second CARs can have an antigen binding domain of a first CAR, such as a fragmented form, such as an scFv, which is not associated with an antigen binding domain of a second CAR, eg, the second CAR The antigen binding domain is VHH.

In some embodiments, the antigen binding domain comprises a single domain antigen binding (SDAB) molecule comprising a molecule wherein the complementarity determining region is part of a single domain polypeptide. Examples include, but are not limited to, heavy chain variable domains, binding molecules that naturally lack light chains, single domains derived from conventional 4-chain antibodies, engineered domains, and sequences other than those derived from antibodies Domain skeleton. The SDAB molecule can be any of the techniques, or any future single domain molecule. SDAB molecules can be derived from any species, including but not limited to mice, humans Classes, camels, llamas, scorpions, fish, sharks, goats, rabbits and cattle. The term also encompasses naturally occurring single domain antibody molecules from species other than camels and sharks.

In one aspect, the SDAB molecule can be derived from a variable region of an immunoglobulin found in fish, such as an immunoglobulin isotype derived from a novel antigen receptor (NAR) found in shark serum. Methods for generating single domain molecules derived from the variable regions of NAR ("IgNAR") are described in WO 03/014161 and Streltsov (2005) Protein Sci. 14:2901-2909.

According to another aspect, the SDAB molecule is a naturally occurring single domain antigen binding molecule, which is referred to as a heavy chain lacking a light chain. Such single domain molecules are disclosed, for example, in WO 9404678 and Hamers-Casterman, C. et al., (1993) Nature 363:446-448. For clarity, this variable domain derived from a heavy chain molecule that naturally lacks a light chain is referred to herein as a VHH or nanobody to distinguish it from the conventional VH of a four-chain immunoglobulin. Such VHH molecules can be derived from camelid species such as camels, llamas, dromedaries, alpacas and guanaco. Other species than camelids can produce heavy chain molecules that naturally lack light chains; such VHHs are within the scope of the invention.

SDAB molecules can be recombinant, CDR-grafted, humanized, camelized, de-immunized, and/or produced in vitro (eg, by phage display selection).

It has also been discovered that cells having a plurality of chimeric membrane-embedded receptors (including antigen binding domains that interact between the antigen binding domains of such receptors) may be undesirable as they inhibit the antigen binding domain The ability of one or more to bind to its cognate antigen. Thus, disclosed herein are cells having first and second non-naturally occurring chimeric membrane-embedded receptors, including antigen binding domains that minimize such interactions. Also disclosed herein are nucleic acids encoding first and second non-naturally occurring chimeric membrane-embedded receptors (including antigen binding domains that minimize such interactions), and methods of making and using such cells and nucleic acids . In one embodiment, the antigen binding domain of one of the first and second non-naturally occurring chimeric membrane-embedded receptors comprises an scFv and the other comprises a single A VH domain, such as a camelid family, a shark or a seven-single VH domain, or a single VH domain derived from a human or mouse sequence.

In some embodiments, the invention encompasses first and second CARs, wherein the antigen binding domain of one of the first CAR and the second CAR does not comprise a variable light chain domain and a variable heavy chain domain . In some embodiments, the antigen binding domain of one of the first CAR and the second CAR is an scFv and the other is not an scFv. In some embodiments, the antigen binding domain of one of the first CAR and the second CAR comprises a single VH domain, such as a camelid, shark or seven-single VH domain, or derived from human or small A single VH domain of a mouse sequence. In some embodiments, the antigen binding domain of one of the first CAR and the second CAR comprises a nanobody. In some embodiments, the antigen binding domain of one of the first CAR and the second CAR comprises a camelid VHH domain.

In some embodiments, the antigen binding domain of one of the first CAR and the second CAR comprises a scFv and the other comprises a single VH domain, such as a camelid, shark or seven-single VH domain Or a single VH domain derived from a human or mouse sequence. In some embodiments, the antigen binding domain of one of the first CAR and the second CAR comprises an scFv and the other comprises a nanobody. In some embodiments, the antigen binding domain of one of the first CAR and the second CAR comprises an scFv and the other comprises a camelid VHH domain.

In some embodiments, the binding of the antigen binding domain of the first CAR to its cognate antigen is not substantially reduced by the presence of the second CAR when present on the cell surface. In some embodiments, the binding of the antigen binding domain of the first CAR to its cognate antigen in the presence of the second CAR is the antigen binding domain of the first CAR and its cognate antigen in the absence of the second CAR 85%, 90%, 95%, 96%, 97%, 98% or 99% of the combination.

In some embodiments, when present on a cell surface, the antigen-binding domains of the first and second CARs are associated with each other less than the case where both are scFv antigen binding domains. In some embodiments, the first CAR and the antigen binding domain of the second CAR are in each other The association is less than 85%, 90%, 95%, 96%, 97%, 98% or 99% of the case where both are scFv antigen binding domains.

In another aspect, the CAR-expressing cells described herein can further express another agent, such as an agent that enhances the activity of cells expressing CAR. For example, in one embodiment, the agent can be an agent that inhibits the inhibitory molecule, such as an agent described herein. In some embodiments, an inhibitory molecule (eg, PD1) can reduce the ability of a cell that exhibits CAR to establish an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (eg, CEACAM-1, CEACAM-3, and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine and TGFR beta. In one embodiment, the agent that inhibits the inhibitory molecule comprises a first polypeptide (eg, an inhibitory molecule) that is associated with a second polypeptide that provides a positive signal to the cell (eg, an intracellular signaling domain as described herein). In one embodiment, the agent comprises, for example, a first polypeptide of an inhibitory molecule, such as PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (eg, CEACAM-1, CEACAM-3, and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II , GAL9, adenosine and TGFR β, or a fragment of any of these (eg, at least a portion of the extracellular domain of any of these); and an intracellular signaling domain as described herein (eg, comprising a common A second polypeptide that stimulates a domain (such as, for example, 41BB, CD27, ICOS, or CD28 as described herein) and/or a major signaling domain (eg, a CD3ζ signaling domain as described herein). In one embodiment, the agent comprises a first polypeptide of PD1 or a fragment thereof (eg, at least a portion of an extracellular domain of PD1), and a second polypeptide of an intracellular signaling domain described herein (eg, as described herein) The CD28 signaling domain and/or the CD3ζ signaling domain described herein). In an embodiment, this The cells of the expression CAR described herein comprise a switch co-stimulatory receptor, as described, for example, in WO 2013/019615, which is incorporated herein by reference in its entirety. PD1 is an inhibitory member of the CD28 receptor family, which also includes CD28, CTLA-4, ICOS, and BTLA. PD-1 is expressed on activated B cells, T cells, and bone marrow cells (Agata et al. 1996 Int. Immunol 8: 765-75). Two ligands of PD1, PD-L1 and PD-L2, have been shown to downregulate T cell activation upon binding to PD1 (Freeman et al, 2000 J Exp Med 192:1027-34; Latchman et al. 2001 Nat Immunol 2:261- 8; Carter et al. 2002 Eur J Immunol 32:634-43). PD-L1 is abundantly present in human cancers (Dong et al. 2003 J Mol Med 81: 281-7; Blank et al. 2005 Cancer Immunol. Immunother 54: 307-314; Konishi et al. 2004 Clin Cancer Res 10: 5094). Immunosuppression can be reversed by inhibiting the local interaction of PD1 with PD-L1.

In one embodiment, the agent comprises an extracellular domain (ECD) of an inhibitory molecule (eg, stylized death 1 (PD1)) that can be fused to a transmembrane domain and an intracellular signaling domain, such as 41BB and CD3ζ (at Also referred to herein as PD1 CAR). In one embodiment, PD1 CAR enhances the persistence of cells expressing CAR, such as T cells or NK cells, when used in combination with BCMA CAR as described herein. In one embodiment, the CAR is a PD1 CAR comprising the extracellular domain of PD1 as indicated by the underline in SEQ ID NO:24. In one embodiment, the PD1 CAR comprises the amino acid sequence of SEQ ID NO: 24.

(SEQ ID NO: 24).

In one embodiment, the PD1 CAR comprises an amino acid sequence (SEQ ID NO: 22) provided below.

(SEQ ID NO: 22).

In one embodiment, the agent comprises a nucleic acid sequence encoding a PD1 CAR (eg, PD1 CAR as described herein). In one embodiment, the nucleic acid sequence of PD1 CAR is shown below, wherein the PD1 ECD is underlined in SEQ ID NO: 23 hereinafter. (SEQ ID NO: 23).

In another aspect, the invention provides a population of cells expressing CAR (eg, CART cells or NK cells expressing CAR). In some embodiments, the population of cells expressing CAR comprises a mixture of cells that exhibit different CARs. For example, in one embodiment, a population of cells expressing CAR (eg, CART cells or NK cells expressing CAR) can include a first cell that exhibits a CAR having an anti-BCMA binding domain as described herein, and exhibits A second cell of CAR of a different anti-BCMA binding domain (eg, an anti-BCMA binding domain as described herein that differs from the anti-BCMA binding domain in the CAR exhibited by the first cell). As another example, a population of cells expressing CAR can include a first cell that exhibits a CAR comprising, for example, an anti-BCMA binding domain as described herein, and the performance includes A second cell of the CAR of the antigen binding domain of a target other than BCMA (eg, CD19, CD20, CS-1, kappa light chain, CD139, Lewis Y antigen, or CD38). In one embodiment, the population of cells expressing CAR comprises a first cell that exhibits an CAR comprising an anti-BCMA binding domain (eg, as described herein) and a CAR that exhibits an antigen binding domain that targets CD19 (CD19 CAR) The second cell. In one embodiment, the population of cells expressing CAR comprises, for example, a first cell that exhibits a CAR comprising a major intracellular signaling domain and a second cell that exhibits a CAR comprising a secondary signaling domain.

In another aspect, the invention provides a population of cells, wherein at least one cell of the population exhibits a CAR having an anti-BCMA binding domain as described herein, and the second cell exhibits another agent, eg, enhanced performance An agent that activates the cells of CAR. For example, in one embodiment, the agent can be an agent that inhibits the inhibitory molecule. In some embodiments, an inhibitory molecule can, for example, reduce the ability of a cell expressing CAR to establish an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (eg, CEACAM-1, CEACAM-3, and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine and TGFR beta. In one embodiment, the agent that inhibits the inhibitory molecule comprises a first polypeptide (eg, an inhibitory molecule) that is associated with a second polypeptide that provides a positive signal to the cell (eg, an intracellular signaling domain as described herein). In one embodiment, the agent comprises, for example, a first polypeptide of an inhibitory molecule, such as PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (eg, CEACAM-1, CEACAM-3, and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II , GAL9, adenosine and TGFR β, or a fragment of any of these (eg, at least a portion of an extracellular domain of any of these); and as described herein Intracellular signaling domain (eg, comprising a costimulatory domain (eg, 41BB, CD27, ICOS, or CD28 as described herein) and/or a primary signaling domain (eg, a CD3ζ signaling domain as described herein) The second polypeptide. In one embodiment, the agent comprises a first polypeptide of PD1 or a fragment thereof (eg, at least a portion of the extracellular domain of PD1), and an intracellular signaling domain described herein (eg, a CD28 signaling domain as described herein) And/or a second polypeptide of the CD3ζ signaling domain described herein.

In one aspect, the invention provides a mixture comprising a cell population that expresses a CAR (eg, a CART cell or a NK cell that expresses a CAR), such as a mixture of cells that exhibit different CARs, and another agent (eg, a kinase inhibitor, such as herein) A method of combining a kinase inhibitor). In another aspect, the invention provides a population comprising a committed cell (wherein at least one cell of the population exhibits an anti-cancer-associated antigen binding domain as described herein, and the second cell exhibits another agent, For example, a method of enhancing the combination of an agent that exhibits the activity of a cell of a CAR with another agent, such as a kinase inhibitor, such as a kinase inhibitor described herein.

Natural killer cell receptor (NKR) CAR

In one embodiment, the CAR molecule described herein comprises one or more components of a natural killer cell receptor (NKR), thereby forming an NKR-CAR. The NKR component can be a transmembrane domain, a hinge domain or a cytoplasmic domain from any of the following natural killer cell receptors: killer cell immunoglobulin-like receptor (KIR), such as KIR2DL1, KIR2DL2/L3, KIR2DL4, KIR2DL5A , KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, DIR2DS5, KIR3DL1/S1, KIR3DL2, KIR3DL3, KIR2DP1 and KIR3DP1; natural cytotoxic receptors (NCR), such as NKp30, NKp44, NKp46; signaling cells of immune cell receptors Activating molecule (SLAM) family, such as CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME and CD2F-10; Fc receptors (FcR), such as CD16 and CD64; and Ly49 receptors, such as LY49A, LY49C. The NKR-CAR molecule described in this article can be used with A molecular or intracellular signaling domain, such as a DAP12 interaction. An exemplary configuration and sequence of a CAR molecule comprising a NKR component is described in International Publication No. WO 2014/145252, the disclosure of which is incorporated herein by reference.

Strategy for the regulation of chimeric antigen receptors

There are many ways to regulate CAR activity. In some embodiments, a regulatable CAR (RCAR) that controls CAR activity is required to optimize the safety and efficacy of CAR therapy. For example, apoptosis is induced using, for example, caspase fused to a dimeric domain (see, for example, Di et al, N Egnl. J. Med. November 3, 2011; 365(18): 1673-1683) It can be used as a safety switch for the CAR therapy of the present invention. In another example, a cell expressing CAR can also exhibit an inducible caspase-9 molecule that is administered a dimeric drug (eg, rimiducid (also known as AP1903). (Bellicum Pharmaceuticals) or AP20187 (Ariad) causes activation of caspase-9 and apoptosis of such cells. The iCaspase-9 molecule contains a dimeric dimeric (CID) binding domain that mediates dimerization in the presence of CID. a chemoducing agent. This results in the inducibility and selective deletion of cells expressing CAR. In some cases, the iCaspase-9 molecule is encoded by a nucleic acid molecule separate from the CAR-encoding vector. In some cases, the iCaspase-9 molecule is The nucleic acid molecule is identical to the CAR-encoding vector. iCaspase-9 provides a safety switch to avoid any toxicity of cells expressing CAR. See, for example, Song et al. Cancer Gene Ther. 2008; 15(10): 667-75; Clinical Trial Id. No. NCT02107963; and Di Stasi et al. N. Engl. J. Med. 2011; 365: 1673-83.

Alternative strategies for modulating CAR therapies of the invention include the use of small molecules that activate or cleave CAR activity, for example, by deleting cells expressing CAR, for example by inducing antibody-dependent cell-mediated cytotoxicity (ADCC) Or antibody. For example, a CAR-expressing cell described herein can also exhibit an antigen recognized by a molecule capable of inducing cell death, such as ADCC or complement-induced cell death. For example, a CAR-expressing cell described herein can also be a receptor that can be targeted by an antibody or antibody fragment. Such receptors Examples include EpCAM, VEGFR, integrins (eg, integrin ανβ3, α4, αI3⁄4β3, α4β7, α5β1, ανβ3, αν), members of the TNF receptor superfamily (eg TRAIL-R1, TRAIL-R2), PDGF receptor, interference Receptor, folate receptor, GPNMB, ICAM-1, HLA-DR, CEA, CA-125, MUC1, TAG-72, IL-6 receptor, 5T4, GD2, GD3, CD2, CD3, CD4, CD5, CD11, CD11a/LFA-1, CD15, CD18/ITGB2, CD19, CD20, CD22, CD23/lgE receptor, CD25, CD28, CD30, CD33, CD38, CD40, CD41, CD44, CD51, CD52, CD62L, CD74, CD80, CD125, CD147/basal immunoglobulin, CD152/CTLA-4, CD154/CD40L, CD195/CCR5, CD319/SLAMF7 and EGFR, and truncated versions thereof (eg, maintaining one or more extracellular epitopes) However, it lacks the pattern of one or more regions within the cytoplasmic domain). For example, the CAR-expressing cells described herein may also exhibit a lack of signaling but retain the truncation of epitopes recognized by molecules capable of inducing ADCC, such as cetuximab (ERBITUX®). Epidermal growth factor receptor (EGFR), which causes the administration of cetuximab to induce ADCC and subsequently express CAR cell deletion (see, for example, WO2011/056894, and Jonnalagadda et al, Gene Ther. 2013; 20(8) 853- 860). Another strategy involves the expression of a highly tight marker/suicide gene that binds to a target epitope of CD32 and CD20 antigen in a cell expressing CAR as described herein, which binds to rituximab, for example by ADCC. Selective deletion of cells expressing CAR (see, eg, Philip et al., Blood. 2014; 124(8) 1277-1287). Other methods for the deletion of cells expressing CAR as described herein include administration of CAMPATH®, a monoclonal anti-CD52 antibody that selectively binds and targets mature lymphocytes, such as cells expressing CAR. For example, by inducing ADCC for destruction. In other embodiments, cells expressing CAR can be selectively targeted using a CAR ligand, such as an anti-idiotypic antibody. In some embodiments, an anti-idiotypic antibody can elicit an effector cell activity, such as ADCC or ADC activity, thus reducing the number of cells expressing CAR. In other embodiments, a CAR ligand, such as an antibody Individual genotype antibodies can be coupled to agents that induce cell killing, such as toxins, thereby reducing the number of cells expressing CAR. Alternatively, the CAR molecule itself can be configured to modulate activity, such as opening and cutting, as described below.

In some embodiments, the RCAR comprises a set of polypeptides, typically two in the simplest embodiment, wherein the components of the standard CAR described herein (eg, antigen binding domain and intracellular signaling domain) are assigned to separate On a peptide or member. In some embodiments, the set of polypeptides comprises a dimeric switch that, in the presence of a dimeric molecule, can couple the polypeptides to each other, for example, to couple an antigen binding domain to an intracellular signaling domain. Additional descriptions and illustrative configurations of such regulatable CARs are provided herein and in International Publication No. WO 2015/090229, which is incorporated herein by reference in its entirety.

In one embodiment, the RCAR comprises two polypeptides or members: 1) an intracellular signaling member comprising an intracellular signaling domain (eg, a major intracellular signaling domain as described herein) and a first switch domain 2) an antigen binding member comprising an antigen binding domain as described herein (eg, targeting a tumor antigen as described herein) and a second switch domain. Where appropriate, RCAR comprises a transmembrane domain as described herein. In an embodiment, the transmembrane domain can be disposed on an intracellular signaling member, on an antigen binding member, or both. (Unless otherwise indicated, when a member or element of RCAR is described herein, the order may be as provided, but may also include other sequences. In other words, in one embodiment, the order is as stated in the text, but in other embodiments, The order may vary. For example, the order of the elements on one side of the transmembrane region may differ from the example, such as the position of the switch domain relative to the intracellular signaling domain, such as upside down.

In an embodiment, the first and second switch domains can form an intracellular or extracellular dimeric switch. In an embodiment, the dimerization switch can be a homodimeric switch, such as where the first and second switch domains are the same, or a heterodimeric switch, such as where the first and second switch domains are different from one another.

In an embodiment, the RCAR may include a "multiple switch." Multiple switches can contain miscellaneous A polyswitch domain or a homodimeric switch domain. Multiple switches independently comprise a plurality (eg, 2, 3, 4, 5, 6, 7, 8, 9, or 10) on a first member (eg, an antigen binding member) and a second member (eg, an intracellular signaling member) Switch domain. In an embodiment, the first member may comprise a plurality of first switch domains, such as a FKBP based switch domain, and the second member may comprise a plurality of second switch domains, such as a FRB based switch domain. In an embodiment, the first member may include first and second switch domains, such as a FKBP-based switch domain and an FRB-based switch domain, and the second member may include first and second switch domains, For example, a switch domain based on FKBP and a switch domain based on FRB.

In one embodiment, the intracellular signaling member comprises one or more intracellular signaling domains, such as a major intracellular signaling domain, and one or more co-stimulatory signaling domains.

In one embodiment, an antigen binding member can comprise one or more intracellular signaling domains, such as one or more co-stimulatory signaling domains. In one embodiment, the antigen binding member comprises a plurality (eg, 2 or 3) of the costimulatory signaling domains described herein, eg, selected from the group consisting of 4-1BB, CD28, CD27, ICOS, and OX40, and in embodiments There is no major intracellular signaling domain. In one embodiment, the antigen binding member comprises the following co-stimulatory signaling domain from the extracellular to the intracellular direction: 4-1BB-CD27; 4-1BB-CD27; CD27-4-1BB; 4-1BB-CD28; CD28 -4-1BB; OX40-CD28; CD28-OX40; CD28-4-1BB; or 4-1BB-CD28. In such embodiments, the intracellular binding member comprises a CD3ζ domain. In one such embodiment, the RCAR comprises (1) an antigen binding member comprising, for example, an antigen binding domain, a transmembrane domain and two co-stimulatory domains and a first switch domain; and (2) an intracellular signal A transduction domain comprising a transmembrane domain or a membrane plug domain and at least one major intracellular signaling domain, and a second switch domain.

One embodiment provides a RCAR in which an antigen binding member is not tethered to a CAR cell surface. This allows cells with intracellular signaling members to conveniently pair with one or more antigen binding domains without transforming the cells with sequences encoding the antigen binding members. In such embodiments, the RCAR comprises: 1) an intracellular signaling member comprising: a first switch domain, a transmembrane domain, an intracellular signaling domain (eg, a major intracellular signaling domain), and a switch domain; and 2) an antigen binding member comprising: an antigen binding domain and a second switch domain, wherein the antigen binding member does not comprise a transmembrane domain or a membrane plug domain, and optionally does not comprise a cell Internal signaling domain. In some embodiments, the RCAR can further comprise 3) a second antigen binding member comprising: a second antigen binding domain, eg, a second antigen binding domain that binds to an antigen different from the antigen binding domain; Two switch domains.

Also provided herein is RCAR, wherein the antigen binding member comprises bispecific activation and targeting capabilities. In this embodiment, the antigen binding member may comprise a plurality (eg, 2, 3, 4, or 5) of antigen binding domains (eg, scFv), wherein each antigen binding domain binds to a target antigen, eg, a different antigen or the same Antigens, such as the same or different epitopes of the same antigen. In one embodiment, a plurality of antigen binding domains are ligated in series, and a linker or hinge region is disposed between each antigen binding domain as appropriate. This document describes suitable connectors and hinge regions.

One embodiment provides an RCAR with a configuration that allows for switch propagation. In this embodiment, the RCAR comprises: 1) an intracellular signaling member comprising: a transmembrane domain or a membrane plug domain as appropriate; one or more co-stimulatory signaling domains, eg, selected from 4 -1BB, CD28, CD27, ICOS and OX40, and a switch domain; and 2) an antigen binding member comprising: an antigen binding domain, a transmembrane domain, and a major intracellular signaling domain, such as a CD3ζ domain, wherein The antigen binding member does not comprise a switch domain or does not comprise a switch domain that dimerizes with a switch domain on an intracellular signaling member. In one embodiment, the antigen binding member does not comprise a costimulatory signaling domain. In an embodiment, the intracellular signaling member comprises a switch domain from a homodimeric switch. In a In an embodiment, the intracellular signaling member comprises a first switch domain of a heterodimeric switch and the RCAR comprises a second intracellular signaling member comprising a second switch domain of a heterodimeric switch. In such embodiments, the second intracellular signaling member comprises the same intracellular signaling domain as the intracellular signaling member. In an embodiment, the dimerization switch is intracellular. In an embodiment, the dimerization switch is extracellular.

In any of the RCAR configurations described herein, the first and second switch domains comprise FKBP/FRB based switches as described herein.

Also provided herein are cells comprising the RCARs described herein. Any cell engineered to express RCAR can be used as a RCARX cell. In one embodiment, the RCARX cells are T cells and are referred to as RCART cells. In one embodiment, the RCARX cells are NK cells and are referred to as RCARN cells.

Nucleic acids and vectors comprising the RCAR coding sequence are also provided herein. The sequences encoding the various elements of RCAR can be placed on the same nucleic acid molecule, such as the same plastid or vector, such as a viral vector, such as a lentiviral vector. In one embodiment, (i) a sequence encoding an antigen binding member and (ii) a sequence encoding an intracellular signaling member may be present on the same nucleic acid, such as a vector. The corresponding protein can be produced, for example, by using a separate promoter or by using a bicistronic transcript that can be produced by cleavage of a single translation product or by translation of two separate protein products. In one embodiment, a sequence encoding a cleavable peptide (eg, a P2A or F2A sequence) is disposed between (i) and (ii). In an embodiment, the sequence encoding the IRES (eg, EMCV or EV71 IRES) is disposed between (i) and (ii). In these embodiments, (i) and (ii) are transcribed in the form of a single RNA. In one embodiment, the first promoter is operably linked to (i) and the second promoter is operably linked to (ii) such that (i) and (ii) are transcribed in separate mRNA formats.

Alternatively, sequences encoding various elements of RCAR can be placed on different nucleic acid molecules, such as different plastids or vectors, such as viral vectors, such as lentiviral vectors. For example, (i) a sequence encoding an antigen binding member can be present on a first nucleic acid (eg, a first vector), and (ii) A sequence encoding an intracellular signaling member can be present on a second nucleic acid (eg, a second vector).

Dimerization switch

The dimerization switch can be non-covalent or covalent. In non-covalent dimeric switches, dimeric molecules promote non-covalent interactions between the switch domains. In covalent dimeric switches, dimeric molecules promote covalent interactions between the switch domains.

In an embodiment, the RCAR comprises a dimeric switch based on FKBP/FRAP or FKBP/FRB. FKBP12 (FKBP or FK506 binding protein) is a rich cytoplasmic protein that acts as an initial intracellular target for the natural product immunosuppressive drug (rapamycin). Rapamycin binds to FKBP and the large PI3K homolog FRAP (RAFT, mTOR). FRB is the 93 amino acid moiety of FRAP, which is sufficient for binding to FKBP-rapamycin complex (Chen, J., Zheng, XF, Brown, EJ & Schreiber, SL (1995) Identification of an 11-kDa FKBP12 - rapamycin-binding domain within the 289-kDa FKBP12-rapamycin-associated protein and characterization of a critical serine residue. Proc Natl Acad Sci USA 92:4947-51).

In an embodiment, a switch based on FKBP/FRAP (eg, FKBP/FRB) can use a dimeric molecule such as rapamycin or a rapamycin analog.

The amino acid sequence of FKBP is as follows: (SEQ ID NO: 275)

In embodiments, the FKBP switch domain can comprise an FKBP fragment capable of binding to FRB, or a fragment or analog thereof, in the presence of a rapamycin or rapamycin analogue, such as the underlined portion of SEQ ID NO: 275, as follows: (SEQ ID NO: 276)

The amino acid sequence of FRB is as follows: ILWHEMWHEG LEEASRLYFG ERNVKGMFEV LEPLHAMMER GPQTLKETSF NQAYGRDLME AQEWCRKYMK SGNVKDLTQA WDLYYHVFRR ISK (SEQ ID NO: 277)

"FKBP/FRAP, eg, a switch of FKBP/FRB" as used herein, refers to a dimeric switch comprising: a first switch domain comprising a molecule capable of resembling rapamycin or rapamycin An FKBP fragment or an analog thereof that binds FRB or a fragment or analog thereof in the presence of a substance (eg, RAD001) and has at least 70%, 75%, 80%, 85%, 90 with the FKBP sequence of SEQ ID NO: 275 or 276 %, 95%, 96%, 97%, 98% or 99% identity or difference is no more than 30, 25, 20, 15, 10, 5, 4, 3, 2 or 1 amino acid residues And a second switch domain comprising a FRB fragment or analog thereof capable of binding to FRB or a fragment or analog thereof in the presence of a rapamycin or rapamycin analogue, and to the FRB of SEQ ID NO:277 The sequence has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity or differs in no more than 30, 25, 20, 15, 10 , 5, 4, 3, 2 or 1 amino acid residue. In one embodiment, the RCAR described herein comprises a switch domain comprising an amino acid residue as disclosed in SEQ ID NO: 275 (or SEQ ID NO: 276), and comprising the SEQ ID NO: 277 A switch domain of the amino acid residue is disclosed.

In an embodiment, the FKBP/FRB dimerization switch comprises a modified FRB switch domain that exhibits a modified (eg, enhanced) FRB-based switch domain, such as a modified FRB switch domain, a FKBP-based switch domain Complex formation with dimeric molecules such as rapamycin or raparog, such as RAD001. In one embodiment, the modified FRB switch domain comprises one or more mutations, eg, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more selected from the group consisting of amino acid positions L2031 Mutations at E2032, S2035, R2036, F2039, G2040, T2098, W2101, D2102, Y2105, and F2108, wherein the wild-type amino acid is mutated to any other naturally occurring amino acid. In a In the examples, the mutant FRB comprises a mutation at E2032, wherein E2032 is mutated to phenylalanine (E2032F), methionine (E2032M), arginine (E2032R), proline (E2032V), tyrosine (E2032Y) Isoleucine (E2032I), for example SEQ ID NO: 278, or leucine (E2032L), for example SEQ ID NO: 279. In one embodiment, the mutant FRB comprises a mutation at T2098, wherein T2098 is mutated to phenylalanine (T2098F) or leucine (T2098L), such as SEQ ID NO: 280. In one embodiment, the mutant FRB comprises a mutation at E2032 and T2098, wherein E2032 is mutated to any amino acid, and wherein T2098 is mutated to any amino acid, such as SEQ ID NO:281. In one embodiment, the mutated FRB comprises an E2032I and T2098L mutation, such as SEQ ID NO: 282. In one embodiment, the mutated FRB comprises an E2032L and T2098L mutation, such as SEQ ID NO: 283.

Other suitable dimerization switches include GyrB-GyrB based dimerization switches, gibberellin based dimerization switches, label/adhesive dimerization switches, and halogen-tag/snap-tag dimerization switches. Such switches and related dimeric molecules will be apparent to those of ordinary skill in the art in light of the teachings provided herein.

Dimeric molecule

Dimeric molecules promote association between switch domains. The presence of an association between dimeric molecular interactions or switch domains allows for association (eg, fusion) with the first switch domain Signal transduction between a polypeptide and a polypeptide associated with (eg, fused to) a second switch domain. As measured, for example, in the systems described herein, signal transduction increases by 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 5 in the presence of non-limiting levels of dimeric molecules. 10, 50, 100 times.

Rapamycin and rapamycin analogues (sometimes referred to as Reparog), such as RAD001, can be used as dimeric molecules in the FKBP/FRB based dimeric switches described herein. In one embodiment, the dimeric molecule can be selected from the group consisting of rapamycin (sirolimus), RAD001 (everolimus), zotarolimus, and temsirolimus ( Temsirolimus), AP-23573 (ridaforolimus), biolimus and AP21967. Additional rapamycin analogues suitable for use with FKBP/FRB based dimerization switches are further described in the subsection entitled "Combination Therapy" or in the subsection entitled "Combination with Low Immunopotentiating Dosage mTOR Inhibitors" .

Split CAR

In some embodiments, cells expressing CAR use a dividing CAR. The split CAR method is described in more detail in the publications WO 2014/055442 and WO 2014/055657, which are incorporated herein by reference. Briefly, a split CAR system comprises a cell that exhibits a first CAR having a first antigen binding domain and a costimulatory domain (eg, 41BB), and the cell also exhibits a second antigen binding domain and an intracellular signaling domain. The second CAR (eg CD3ξ). When the cell encounters the first antigen, the co-stimulatory domain is activated and the cell proliferates. When the cell encounters a second antigen, it activates the intracellular signaling domain and initiates cell killing activity. Thus, cells expressing CAR are fully activated only in the presence of both antigens. In an embodiment, the first antigen binding domain recognizes BCMA, eg, comprising an antigen binding domain as described herein, and the second antigen binding domain recognizes an antigen expressed on acute myeloid leukemia cells, eg, CD123, CLL-1 , CD34, FLT3 or folate receptor beta. In an embodiment, the first antigen binding domain recognizes BCMA, eg, as described herein An antigen binding domain, and the second antigen binding domain recognizes an antigen expressed on a B cell, such as CD10, CD19, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b or CD79a.

Stability and mutation

The stability of an anti-BCMA binding domain, such as a scFv molecule (e.g., soluble scFv), can be assessed by reference to the biophysical properties (e.g., thermostability) of a conventional control scFv molecule or full length antibody. In one embodiment, the thermostability of the humanized scFv in the assay is about 0.1, about 0.25, about 0.5, about 0.75, about 1, about 1.25, about 1.5 higher than the control binding molecule (eg, a conventional scFv molecule). , about 1.75, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10 degrees Celsius, about 11 degrees Celsius, about 12 degrees Celsius, about 13 degrees Celsius, about 14 degrees Celsius, or about 15 degrees Celsius.

Subsequent administration of improved thermal stability of the entire CART-BCMA construct with an anti-BCMA binding domain (e.g., scFv) results in improved therapeutic properties of the CART-BCMA construct. The thermal stability of the anti-BCMA binding domain (e.g., scFv) can be increased by at least about 2 °C or 3 °C as compared to conventional antibodies. In one embodiment, the thermal stability of the anti-BCMA binding domain (eg, scFv) is increased by 1 °C compared to conventional antibodies. In another embodiment, the thermostability of the anti-BCMA binding domain (eg, scFv) is increased by 2 °C compared to conventional antibodies. In another embodiment, the thermal stability of the scFv is increased by 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 °C compared to conventional antibodies. Comparisons can be made, for example, between the scFv molecules disclosed herein and the scFv molecules or Fab fragments of the antibodies from which the scFv VH and VL are derived. Thermal stability can be measured using methods known in the art. For example, in one embodiment, Tm can be measured. Methods for measuring Tm and other methods for determining protein stability are described in more detail below.

Mutations in scFv (induced by humanization or direct mutation of soluble scFv) alter the stability of the scFv and increase the overall stability of the scFv and CART33 constructs. Can be used The stability of human scFv and murine scFv was compared between the measured values of Tm, denaturation temperature and aggregation temperature.

The binding ability of the mutant scFv can be determined using the assay described in the examples.

In one embodiment, the anti-BCMA binding domain (eg, scFv) comprises at least one mutation caused by a humanization method such that the mutated scFv confers stability to the CART-BCMA construct. In another embodiment, the anti-BCMA binding domain (eg, scFv) comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mutations caused by the humanization method such that the mutation The scFv confers improved stability to the CART-BCMA construct.

Method for assessing protein stability

The stability of the antigen binding domain can be assessed using, for example, the methods described below. Such methods allow for the determination of multiple thermal expansion transitions, where the most unstable domain first unfolds or limits the overall stability threshold of the co-expanded multi-domain unit (eg, a multi-domain protein showing a single unfolded transition). The most unstable domains can be identified in many other ways. Mutation induction can be performed on probes whose domains limit the overall stability. In addition, protease resistance of multidomain proteins can be carried out under conditions in which the most unstable domains are known to be essentially developed by DSC or other spectroscopic methods (Fontana et al., (1997) Fold. Des., 2: R17-26; Dimasi et al. (2009) J. Mol. Biol. 393: 672-692). Once the most unstable domain is identified, the sequence encoding this domain (or a portion thereof) can be used as the test sequence in the method.

a) thermal stability

The thermal stability of the composition can be analyzed using a variety of non-limiting biophysical or biochemical techniques known in the art. In certain embodiments, thermal stability is assessed by analytical spectroscopy.

An exemplary analytical spectroscopy method is Differential Scanning Calorimetry (DSC). DSCs employ a calorimeter that is sensitive to the endotherm associated with most protein or protein domain development (see, for example, Sanchez-Ruiz et al, Biochemistry, 27: 1648-52, 1988). For testing To determine the thermal stability of the protein, the protein sample is inserted into the calorimeter and the temperature is raised until the Fab or scFv is unfolded. The temperature at which the protein is released indicates the overall protein stability.

Another exemplary analytical spectroscopy method is circular dichroism (CD) spectroscopy. CD spectroscopy measures the change in optical activity of the composition as a function of temperature. Circular dichroism (CD) spectroscopy measures the difference in absorption of left-handed polarized light versus right-handed polarized light caused by structural asymmetry. The disordered or unfolded structure produces a CD spectrum that is very different from the ordered or folded structure. The CD spectrum reflects the sensitivity of the protein to increased denaturation of the temperature and thus indicates the thermal stability of the protein (see van Mierlo and Steemsma, J. Biotechnol., 79(3): 281-98, 2000).

Another exemplary analytical spectroscopy for measuring thermal stability is fluorescence emission spectroscopy (see van Mierlo and Steemsma, supra). Yet another exemplary analytical spectroscopy for measuring thermal stability is nuclear magnetic resonance (NMR) spectroscopy (see, for example, Mierlo and Steemsma, supra).

The thermal stability of the composition can be measured biochemically. An exemplary biochemical method for assessing thermal stability is thermal attack analysis. In "Hot Attack Analysis," the composition is subjected to a series of elevated temperatures for a set period of time. For example, in one embodiment, the test scFv molecule or molecule comprising the scFv molecule is subjected to a series of increasing temperatures, such as for 1-1.5 hours. The activity of the protein is then analyzed by relevant biochemical analysis. For example, if the protein is a binding protein (eg, an scFv or a polypeptide containing an scFv), the binding activity of the binding protein can be determined by functional or quantitative ELISA.

Such assays can be performed in high throughput formats and in the formats disclosed in the examples using E. coli and high throughput screening. A library of anti-BCMA binding domain (e.g., scFv) variants can be generated using methods known in the art. An anti-BCMA binding domain (e.g., scFv) can be elicited, and an anti-BCMA binding domain (e.g., scFv) can undergo thermal attack. The binding of the challenged test samples can be analyzed, and the stable anti-BCMA binding domains (eg, scFv) can be scaled up and further characterized.

Measuring the melting of the composition by using any of the above techniques (eg, analytical spectroscopy techniques) Melting temperature (Tm) to evaluate thermal stability. The melting temperature is the temperature at a point in the thermal transition curve in which 50% of the molecules of the composition are in a folded state (see, for example, Dimasi et al. (2009) J. Mol Biol. 393: 672-692). In one embodiment, the Tm value of the anti-BCMA binding domain (eg, scFv) is about 40 ° C, 41 ° C, 42 ° C, 43 ° C, 44 ° C, 45 ° C, 46 ° C, 47 ° C, 48 ° C, 49 ° C. 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66 °C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C , 100 ° C. In one embodiment, the IgG has a Tm value of about 40 ° C, 41 ° C, 42 ° C, 43 ° C, 44 ° C, 45 ° C, 46 ° C, 47 ° C, 48 ° C, 49 ° C, 50 ° C, 51 ° C, 52 ° C. , 53 ° C, 54 ° C, 55 ° C, 56 ° C, 57 ° C, 58 ° C, 59 ° C, 60 ° C, 61 ° C, 62 ° C, 63 ° C, 64 ° C, 65 ° C, 66 ° C, 67 ° C, 68 ° C, 69 °C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86 ° C, 87 ° C, 88 ° C, 89 ° C, 90 ° C, 91 ° C, 92 ° C, 93 ° C, 94 ° C, 95 ° C, 96 ° C, 97 ° C, 98 ° C, 99 ° C, 100 ° C. In one embodiment, the Tm value of the multivalent antibody is about 40 ° C, 41 ° C, 42 ° C, 43 ° C, 44 ° C, 45 ° C, 46 ° C, 47 ° C, 48 ° C, 49 ° C, 50 ° C, 51 ° C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C , 69 ° C, 70 ° C, 71 ° C, 72 ° C, 73 ° C, 74 ° C, 75 ° C, 76 ° C, 77 ° C, 78 ° C, 79 ° C, 80 ° C, 81 ° C, 82 ° C, 83 ° C, 84 ° C, 85 °C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C, 100°C.

Thermal stability is also assessed by measuring the specific heat or heat capacity (Cp) of the composition using analytical calorimetry techniques (e.g., DSC). The specific heat of the composition is the energy required to increase the temperature of 1 mol of water by 1 ° C (e.g., in kcal/mol). Because large Cp is characteristic of denatured or inactivated protein compositions. The change in heat capacity (ΔCp) of the composition was measured by measuring its specific heat before and after the thermal transition of the composition. Thermal stability can also be assessed by measuring or determining other thermodynamic stability parameters, including unfolded Gibbs free energy (ΔG), unfolded 焓 (ΔH), or expanded entropy (ΔS). One or more of the above biochemical analyses (e.g., thermal attack analysis) are used to determine the temperature (i.e., T _C value) at which 50% of the composition retains its activity (e.g., binding activity).

Furthermore, mutations in the anti-BCMA binding domain (eg, scFv) alter the thermostability of the anti-BCMA binding domain (eg, scFv) compared to the unmutated anti-BCMA binding domain (eg, scFv). When a human or humanized anti-BCMA binding domain (eg, scFv) is incorporated into a BCMA construct, an anti-BCMA binding domain (eg, a humanized scFv) confers thermal stability to the entire anti-BCMA CART construct. In one embodiment, the anti-BCMA binding domain (eg, scFv) comprises a single mutation that confers thermostability to the anti-BCMA binding domain (eg, scFv). In another embodiment, the anti-BCMA binding domain (eg, scFv) comprises multiple mutations that confer thermostability to the anti-BCMA binding domain (eg, scFv). In one embodiment, multiple mutations in an anti-BCMA binding domain (eg, scFv) have an additive effect on the thermal stability of a -BCMA binding domain (eg, scFv).

b) Aggregate%

The stability of the composition can be determined by measuring the tendency of its aggregation. Aggregation can be measured by a number of non-limiting biochemical or biophysical techniques. For example, aggregation of the composition can be assessed using chromatography, such as size exclusion chromatography (SEC). SEC separates molecules based on size. The column is filled with semi-solid beads of polymer gel that will allow ions and small molecules to enter the interior but do not permit macromolecules to enter. When the protein composition is applied to the top of the column, the tightly folded proteins (i.e., unaggregated proteins) are distributed in a solvent that is larger than the large protein aggregates. Thus, the large aggregates move through the column more quickly, and in this way, the mixture can be separated or fractionated into its components. Each fraction can be separately quantified (e.g., by light scattering) upon elution from the gel. Thus, the % aggregation of the composition can be determined by comparing the concentration of the dissolved fraction with the total concentration of the protein applied to the gel. Stable composition It is substantially single-dissolved from the column and appears as a substantially single peak in the dissolution profile or chromatogram.

c) binding affinity

The stability of the composition can be assessed by determining its target binding affinity. A variety of methods for determining binding affinity are known in the art. An exemplary method for determining binding affinity employs surface plasmon resonance. Surface plasmon resonance is an optical phenomenon that allows analysis of real-time biospecific interactions by detecting changes in protein concentration in the biosensor matrix using, for example, the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). effect. For further description see Jonsson, U. et al., (1993) Ann. Biol. Clin. 51: 19-26; Jonsson, U., i (1991) Biotechniques 11: 620-627; Johnsson, B. et al., (1995). J. Mol. Recognit. 8: 125-131; and Johnnson, B. et al., (1991) Anal. Biochem. 198: 268-277.

In one aspect, the antigen binding domain of CAR comprises an amino acid sequence homologous to the antigen binding domain amino acid sequence described herein, and the antigen binding domain retains an anti-BCMA antibody fragment described herein. Required features. In one particular aspect, the CAR composition of the invention comprises an antibody fragment. In another aspect, the antibody fragment comprises an scFv.

In various aspects, the antigen binding domain of CAR is modified within one or two variable regions (eg, VH and/or VL), eg, within one or more CDR regions and/or one or more framework regions One or more amino acids are engineered. In one particular aspect, the CAR composition of the invention comprises an antibody fragment. In another aspect, the antibody fragment comprises an scFv.

One of ordinary skill will appreciate that the antibodies or antibody fragments of the invention can be further modified such that their amino acid sequences (e.g., from the wild type) vary, but the desired activity is unchanged. For example, additional nucleotide substitutions can be made to the protein, such as conservative substitutions that result in amino acid substitutions, such as conservative substitutions at "non-essential" amino acid residues. For example, a non-essential amino acid residue in a molecule can be replaced with another amino acid residue from the same side chain family. In another In one embodiment, the amino acid strip may be replaced by a structurally similar strip in the sequence and/or composition of the side chain family members, for example, an amino acid having an amino acid group having a similar side chain may be carried out. A conservative substitution of residue substitutions.

A family of amino acid residues with similar side chains have been defined in the art, including basic side chains (eg, lysine, arginine, histidine), acidic side chains (eg, aspartic acid, glutamine) Acid), without electrode side chains (eg glycine, aspartame, glutamic acid, serine, threonine, tyrosine, cysteine), non-polar side chains (eg propylamine) Acid, valine, leucine, isoleucine, valine, phenylalanine, methionine, tryptophan), beta branched chain side chain (eg sulphate, valine, isalamine) Acid) and aromatic side chains (eg tyrosine, phenylalanine, tryptophan, histidine).

Percent identity in the context of two or more nucleic acid or polypeptide sequences refers to the same degree of two or more sequences. When comparing and aligning in the comparison window or the specified area for maximum correspondence, if one of the following sequence comparison algorithms is used or by manual comparison and visual measurement, if the two sequences have the same percentage of the same amine Acidic acid residue or nucleotide (eg 60% identity over the entire sequence on a specified region or not specified, optionally 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77) %, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, The 94%, 95%, 96%, 97%, 98%, 99% consistency), the two sequences are "substantially consistent." Consistency, as appropriate, exists over a region of at least about 50 nucleotides (or 10 amino acids), or more preferably 100 to 500 or 1000 or more nucleotides (or 20, 50) On the area of the length of 200, more or more amino acids.

For sequence comparison, typically one sequence is used as a reference sequence and the test sequence is compared thereto. When using the sequence comparison algorithm, the test sequence and the reference sequence are input to a computer, subsequence coordinates are designated as necessary, and sequence algorithm program parameters are specified. Preset program parameters can be used, or alternate parameters can be specified. Based on the program parameters, the subsequent sequence comparison algorithm calculates the percent sequence identity of the test sequence relative to the reference sequence. Preamble for comparison Column alignment methods are well known in the art. Optimal sequence alignments can be compared, for example by Smith and Waterman, (1970) Adv. Appl. Math. 2: 482c local homology algorithm, by Needleman and Wunsch, (1970) J. Mol. Biol The homology alignment algorithm of .48:443 is studied by the similarity method of Pearson and Lipman, (1988) Proc. Nat'l. Acad. Sci. USA 85:2444, by computerization of the algorithm Implementation (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package (Genetics Computer Group, 575 Science Dr., Madison, WI)), or by manual comparison and visual inspection (see, for example, Brent et al., (2003) Current Protocols in Molecular Biology).

Two examples of algorithms suitable for determining percent sequence identity and percent sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., (1977) Nuc. Acids Res. 25: 3389-3402; and Altschul, respectively. Et al., (1990) J. Mol. Biol. 215: 403-410. The software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information.

You can also use the algorithm of E. Meyers and W. Miller, (1988) Comput. Appl. Biosci. 4: 11-17 (which has been incorporated into the ALIGN program (version 2.0)), using the PAM120 weight residue table, 12 The gap length penalty and the 4 point gap penalty were used to determine the percent identity between the two amino acid sequences. In addition, the percent identity between the two amino acid sequences can be performed using the algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48: 444-453 (which has been incorporated into the GCG software suite (at www.gcg. The com available in the GAP program), using the Blossom 62 matrix or the PAM250 matrix, and the gap weights of 16, 14, 12, 10, 8, 6, or 4 and the length of 1, 2, 3, 4, 5, or 6 Weights are measured.

In one aspect, the invention encompasses the modification of a starting antibody or fragment (e.g., scFv) amino acid sequence that produces a functionally equivalent molecule. For example, a VH or VL of an anti-BCMA binding domain (eg, scFv) contained in a CAR can be modified to retain at least about 70 of the initial VH or VL framework regions of an anti-BCMA binding domain (eg, scFv). %, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% consistency. The invention encompasses modifications of the entire CAR construct, such as modifications of one or more amino acid sequences of multiple domains of a CAR construct to produce a functionally equivalent molecule. The CAR construct can be modified to retain at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83 %, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% Start CAR structure consistency.

RNA transfection

Methods for generating RNA CAR for in vitro transcription are disclosed herein. The invention also encompasses RNA constructs encoding CAR that can be directly transfected into cells. A method of producing mRNA for transfection can involve the use of a specially designed primer in vitro transcription (IVT) template followed by the addition of poly A to produce 3' and 5' untranslated sequences ("UTR"), 5' capping And/or an internal ribosome entry site (IRES), a nucleic acid to be expressed, and a construct of a poly A tail (SEQ ID NO: 35) of 50-2000 bases in length. The RNA produced can be efficiently transfected into different types of cells. In one aspect, the template includes a sequence of CARs.

In one aspect, the anti-BCMA CAR is encoded by messenger RNA (mRNA). In one aspect, mRNA encoding anti-BCMA CAR is introduced into an immune effector cell, such as a T cell or NK cell, to produce a cell that expresses CAR (eg, a CART cell or a NK cell that expresses CAR).

In one embodiment, the in vitro transcribed RNA CAR can be introduced into the cell in a transiently transfected form. RNA is produced by in vitro transcription using a template generated by polymerase chain reaction (PCR). Relevant DNA from any source can be directly transformed into a template for in vitro mRNA synthesis by PCR using appropriate primers and RNA polymerase. DNA sources can be, for example, genomic DNA, plastid DNA, phage DNA, cDNA, synthetic DNA sequences Or any other suitable source of DNA. The desired template for in vitro transcription is the CAR of the present invention. For example, a template for RNA CAR comprises an extracellular region comprising a single-chain variable domain of an anti-tumor antibody; a hinge region, a transmembrane domain (eg, a transmembrane domain of CD8a); and an intracellular signaling domain (eg, a cytoplasmic region comprising a signaling domain of CD3-ζ and a signaling domain of 4-1BB).

In one embodiment, the DNA to be used in PCR contains an open reading frame. DNA can be derived from naturally occurring DNA sequences of the genome of an organism. In one embodiment, the nucleic acid can include some or all of the 5' and/or 3' untranslated regions (UTRs). Nucleic acids can include exons and introns. In one embodiment, the DNA to be used in PCR is a human nucleic acid sequence. In another embodiment, the DNA to be used in PCR is a human nucleic acid sequence comprising a 5' and 3' UTR. Alternatively, the DNA can be an artificial DNA sequence that is not normally expressed in a naturally occurring organism. An exemplary artificial DNA sequence is a sequence comprising a portion of a gene joined together to form an open reading frame encoding a fusion protein. The DNA portions that are joined together can be from a single organism or more than one organism.

A template for in vitro transcription of mRNA for transfection is generated using PCR. Methods for performing PCR are well known in the art. The primer for PCR is designed to have a region that is substantially complementary to the DNA region to be used as a PCR template. As used herein, "substantially complementary" is a nucleotide sequence in which most or all of the bases in the leader sequence are complementary or one or more bases are not complementary or mismatched. Substantially complementary sequences can be annealed or hybridized to the intended DNA target under annealing conditions for PCR. The primer can be designed to be substantially complementary to any portion of the DNA template. For example, primers can be designed to amplify portions of nucleic acids (open reading frames) that are normally transcribed in cells, including 5' and 3' UTRs. Primers can also be designed to amplify a portion of a nucleic acid encoding a particular domain of interest. In one embodiment, the primers are designed to amplify the coding region of human cDNA, including all or part of the 5' and 3' UTRs. Primers suitable for use in PCR can be produced by synthetic methods well known in the art. A "forward primer" is a nucleoside that contains a nucleotide that is substantially complementary to a nucleotide on a DNA template upstream of the DNA sequence to be amplified. The introduction of the acid zone. "Upstream" is used herein to refer to the 5' position of the DNA sequence to be amplified relative to the encoded strand. A "reverse primer" is an primer containing a nucleotide region substantially complementary to a double-stranded DNA template downstream of a DNA sequence to be amplified. "Downstream" is used herein to refer to the 3' position of the DNA sequence to be amplified relative to the encoded strand.

Any DNA polymerase suitable for use in PCR can be used in the methods disclosed herein. Reagents and polymerases were purchased from a number of sources.

Chemical structures that promote stability and/or translation efficiency can also be used. The RNA preferably has a 5' and 3' UTR. In one embodiment, the 5' UTR is from 1 to 3000 nucleotides in length. The length of the 5' and 3' UTR sequences to be added to the coding region can be varied by various methods including, but not limited to, designing PCR primers that anneal to different regions of the UTR. Using this method, one of ordinary skill can vary the 5' and 3' UTR lengths required to achieve optimal translational efficiency after transfection of transcribed RNA.

The 5' and 3' UTRs can be the naturally occurring endogenous 5' and 3' UTRs of the relevant nucleic acids. Alternatively, the UTR sequences can be added by incorporating a UTR sequence that is not endogenous to the relevant nucleic acid into the forward and reverse primers or by any other modification of the template. The use of a UTR sequence that is not endogenous to the relevant nucleic acid can be adapted to alter the stability and/or translation efficiency of the RNA. For example, AU enrichment elements in the 3' UTR sequence are known to reduce mRNA stability. Thus, the 3' UTR can be selected and designed to increase the stability of the transcribed RNA based on UTR properties well known in the art.

In one embodiment, the 5' UTR may contain a Kozak sequence of an endogenous nucleic acid. Alternatively, when a 5' UTR that is not endogenous to the relevant nucleic acid is added by PCR as described above, the common Czak sequence can be redesigned by adding a 5' UTR sequence. The Kzark sequence improves the efficiency of translation of some RNA transcripts, but does not appear to be required for efficient transfer of all RNA. Many mRNAs are known in the art to require a Kzakh sequence. In other embodiments, the 5' UTR can be the 5' UTR of the RNA virus whose RNA genome is stable in the cell. In other embodiments, multiple nucleotide analogs can be used to block 3' or 5' UTR The exonuclease of mRNA is decomposed.

In order to be able to synthesize RNA from a DNA template without genetic selection, the transcriptional promoter should be ligated to the DNA template upstream of the sequence to be transcribed. When a sequence serving as a promoter of RNA polymerase is added to the 5' end of the forward primer, the RNA polymerase promoter is incorporated into the PCR product upstream of the open reading frame to be transcribed. In a preferred embodiment, the promoter is a T7 polymerase promoter as described elsewhere herein. Other suitable promoters include, but are not limited to, the T3 and SP6 RNA polymerase promoters. Common nucleotide sequences for the T7, T3 and SP6 promoters are known in the art.

In a preferred embodiment, the mRNA has a 5' end capping and a 3' poly (A) tail that determine ribosome binding, translation initiation, and mRNA stability in the cell. On circular DNA templates, such as plastid DNA, RNA polymerase produces long multiplex products that are not suitable for expression in eukaryotic cells. Transcription of linearized plastid DNA at the end of the 3' UTR produces normal-sized mRNA that is ineffective in eukaryotic transfection even after polyadenylation after transcription.

On linear DNA templates, phage T7 RNA polymerase allows the 3' end of the transcript to extend beyond the last base of the template (Schenborn and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal- Herranz, Eur. J. Biochem., 270: 1485-65 (2003)).

A conventional method of integrating a poly A/T elongation into a DNA template is molecular selection. However, poly A/T sequences integrated into plastid DNA can destabilize plastids, which is why plastid DNA templates obtained from bacterial cells are often highly confounded with deletions and other aberrations. This makes the selection process not only laborious and time consuming, but also generally unreliable. This is highly desirable for a method of constructing a DNA template with a poly A/T 3' elongation in the absence of colonization.

A reverse primer containing a poly-T tail (such as 100T tail (SEQ ID NO: 31)) (the size may be 50-5000T (SEQ ID NO: 32)) or by including after PCR may be used during PCR. However, it is not limited to any other method of DNA ligation or in vitro recombination to generate a poly A/T segment of a transcribed DNA template. The poly(A) tail also provides stability to the RNA and reduces its degradation. In general, the length of the poly(A) tail is positively correlated with the stability of the transcribed RNA. In one embodiment, the poly(A) tail is from 100 to 5000 adenosine (SEQ ID NO: 33).

The poly(A) tail of RNA can be further extended after in vitro transcription using a poly(A) polymerase such as E. coli poly A polymerase (E-PAP). In one embodiment, increasing the length of the poly(A) tail from 100 nucleotides to 300 to 400 nucleotides (SEQ ID NO: 34) increases the translation efficiency of the RNA by about two-fold. In addition, the attachment of different chemical groups to the 3' end increases mRNA stability. Such linkages may contain modified/artificial nucleotides, aptamers, and other compounds. For example, an ATP analog can be incorporated into the poly(A) tail using a poly(A) polymerase. ATP analogs can further increase RNA stability.

The 5' capping also provides stability to RNA molecules. In a preferred embodiment, the RNA produced by the methods disclosed herein comprises a 5' capping. 5' capping is provided using techniques known in the art and described herein (Cougot et al, Trends in Biochem. Sci., 29: 436-444 (2001); Stepinski et al, RNA, 7: 1468-95 (2001); Elango et al, Biochim. Biophys. Res. Commun., 330: 958-966 (2005)).

RNA produced by the methods disclosed herein may also contain an internal ribosome entry site (IRES) sequence. The IRES sequence can be any viral, chromosomal or artificially designed sequence that elicits a capsid-independent ribosome that binds to the mRNA and facilitates the initiation of translation. Any lysate suitable for cell electroporation may be included, which may contain factors that contribute to cell permeability and viability, such as sugars, peptides, lipids, proteins, antioxidants, and surfactants.

RNA can be introduced into a target cell using any of a number of different methods including, for example, but not limited to, the following commercially available methods: electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830) (BTX) (Harvard Instruments, Boston, Mass.) or Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendort, Hamburg Germany), cationic liposome transfected with liposome Transfection, polymer encapsulation, peptide-mediated transfection, or bioelastic particle delivery systems, such as "gene guns" (see example) For example, Nishikawa et al., Hum Gene Ther., 12(8): 861-70 (2001)).

Non-viral delivery method

In some aspects, a non-viral method can be used to deliver a nucleic acid encoding a CAR described herein to a cell or tissue or individual.

In some embodiments, the non-viral method involves the use of a transposon (also known as a transposable element). In some embodiments, the transposon is a piece of DNA that can be inserted into the genome itself, such as a DNA piece that is capable of self-replication and insertion of its copy into the genome, or can be spliced from a longer nucleic acid and inserted into the genome. Another piece of DNA. For example, a transposon comprises a DNA sequence consisting of an inverted repeat sequence flanked by a gene for transposition.

Exemplary methods of nucleic acid delivery using transposons include the Sleeping Beauty Transposon System (SBTS) and the piggyBac (PB) transposon system. See, for example, Aronovich et al. Hum. Mol. Genet. 20. R1 (2011): R14-20; Singh et al. Cancer Res. 15 (2008): 2961-2971; Huang et al. Mol. Ther. 16 (2008): 580 - 589; Grabundzija et al. Mol. Ther. 18 (2010): 1200-1209; Kebriaei et al. Blood. 122.21 (2013): 166; Williams. Molecular Therapy 16.9 (2008): 1515-16; Bell et al. Nat. Protoc 2.2.2 (2007): 3153-65; and Ding et al. Cell. 122.3 (2005): 473-83, each of which is incorporated herein by reference.

SBTS consists of two components: 1) a transposon containing the transgene and 2) a transposase source. Transposases can transfer a transposon from a vector plastid (or other donor DNA) to a target DNA, such as a host cell chromosome/genome. For example, a transposase binds to a vector plastid/donor DNA, cleaves a transposon (including a transgene) from a plastid, and inserts it into the genome of the host cell. See, for example, Aronovich et al., supra.

Exemplary transposons include pT2-based transposons. See, for example, Grabundzija et al., Nucleic Acids Res. 41.3 (2013): 1829-47; and Singh et al., Cancer Res. 68.8 (2008): 2961-2971, each incorporated herein by reference. Exemplary transposases include Tc1/hydrogen-type transposases, such as SB10 transposase or SB11 transposase (a highly active Transposase, which can be expressed, for example, from a cytomegalovirus promoter). See, for example, Aronovich et al; Kebriaei et al; and Grabundzija et al., each of which is incorporated herein by reference.

The use of SBTS allows for the efficient integration and performance of a transgene, such as a nucleic acid encoding a CAR as described herein. Provided herein are methods for producing cells, such as T cells or NK cells, that stably exhibit the CARs described herein, for example using a transposon system such as SBTS.

According to the methods described herein, in some embodiments, one or more nucleic acids comprising a SBTS component, such as a plastid, are delivered to a cell (eg, a T or NK cell). For example, a nucleic acid is delivered by standard methods of delivery of a nucleic acid (eg, plastid DNA), such as the methods described herein, such as electroporation, transfection, or lipofection. In some embodiments, the nucleic acid comprises a transposon comprising a transgene, such as a nucleic acid encoding a CAR as described herein. In some embodiments, the nucleic acid comprises a transposon comprising a transgenic gene (eg, a nucleic acid encoding a CAR described herein) and a nucleic acid sequence encoding a transposase. In other embodiments, a system having two nucleic acids is provided, such as a dual plastid system, for example, wherein the first plastid contains a transposon comprising a transgene and the second plastid contains a nucleic acid sequence encoding a transposase. For example, the first and second nucleic acids are co-delivered to a host cell.

In some embodiments, by using a gene that utilizes SBTS for insertion and use of a nuclease (eg, zinc finger nuclease (ZFN), transcriptional activator-like effect nuclease (TALEN), CRISPR/Cas system, or a wide range of engineering A combination of gene editing of the nuclease reengineering homing endonuclease) produces cells expressing the CAR as described herein, such as T or NK cells.

In some embodiments, non-viral delivery methods are used to allow for stylization of cells such as T or NK cells and direct infusion of cells into an individual. Advantages of non-viral vectors include, but are not limited to, easy and relatively low cost production sufficient to satisfy the patient population, stability during storage, and lack of immunogenicity.

Nucleic acid construct encoding CAR

The invention also provides nucleic acid molecules encoding one or more of the CAR constructs described herein. In one aspect, the nucleic acid molecule is provided as a messenger RNA transcript. In one aspect, the nucleic acid molecule is provided as a DNA construct.

Thus, in one aspect, the invention relates to an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an anti-BCMA binding domain (eg, a human anti-BCMA binding domain), Transmembrane domains and intracellular signaling domains comprising a stimulatory domain (eg, a co-stimulatory signaling domain and/or a primary signaling domain, such as a scorpion). In one embodiment, the anti-BCMA binding domain is an anti-BCMA binding domain as described herein, for example comprising selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42. SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: a sequence of a population consisting of SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148 or SEQ ID NO: 149, or An anti-BCMA binding domain with a sequence that is 95%-99% identical. In one embodiment, the transmembrane domain is a transmembrane domain of a protein selected from the group consisting of: alpha, beta or ξ chain of T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8 , CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154. In one embodiment, the transmembrane domain comprises the sequence of SEQ ID NO: 6 or a sequence with 95%-99% identity thereto. In one embodiment, the anti-BCMA binding domain is joined to the transmembrane domain by a hinge region (eg, a hinge as described herein). In one embodiment, the hinge region comprises SEQ ID NO: 2 Or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5, or a sequence with 95%-99% identity thereto. In one embodiment, the isolated nucleic acid molecule further comprises a sequence encoding a costimulatory domain. In one embodiment, the costimulatory domain is a functional signaling domain of a protein selected from the group consisting of: MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, Integrin, signaling lymphocyte activating molecule (SLAM protein), activated NK cell receptor, BTLA, Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA -1 (CD11a/CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB Coordination of -A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a and specific binding to CD83 body. In one embodiment, the costimulatory domain comprises the sequence of SEQ ID NO: 7 or a sequence having 95%-99% identity thereto, or has the sequence of SEQ ID NO: 8 (or is 95%-99% identical thereto) a CD27 costimulatory domain of the sequence of SEQ ID NO: 379, or a CD28 costimulatory domain having a sequence of SEQ ID NO: 379 (or a sequence having 95%-99% identity thereto), or a sequence having SEQ ID NO: 381 The ICOS co-stimulatory domain (or a sequence with 95%-99% identity). in a In the embodiments, the intracellular signaling domain comprises a functional signaling domain of 4-1BB and a functional signaling domain of CD3ξ. In one embodiment, the intracellular signaling domain comprises the sequence of SEQ ID NO:7 or SEQ ID NO:8, or a sequence having 95%-99% identity thereto, and SEQ ID NO:9 or SEQ ID NO A sequence of 10, or a sequence with 95%-99% identity thereof, wherein the sequences comprising the intracellular signaling domain are represented in the same framework and are in the form of a single polypeptide chain.

In another aspect, the invention relates to an isolated nucleic acid molecule encoding a CAR construct comprising a leader sequence of SEQ ID NO: 1, having a sequence selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 40. SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148 or The scFv domain of SEQ ID NO: 149 (or a sequence having 95%-99% identity thereto), SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO :5 (or a sequence with 95%-99% consistency), with S EQ ID NO: a transmembrane domain of a sequence of 6 (or a sequence with 95%-99% identity thereto), a 4-1BB co-stimulatory domain having the sequence of SEQ ID NO: 7 or having SEQ ID NO:8 a CD27 costimulatory domain of the sequence (or a sequence with 95%-99% identity) or a CD28 costimulatory structure having the sequence of SEQ ID NO: 1104 (or a sequence with 95%-99% identity) a domain or an ICOS co-stimulatory domain having the sequence of SEQ ID NO: 1106 (with a sequence having 95%-99% identity), and having CD3 ξ stimulating domain of the sequence of SEQ ID NO: 9 or SEQ ID NO: 10 (or a sequence with 95%-99% identity thereto).

In another aspect, the invention relates to an isolated polypeptide molecule encoded by a nucleic acid molecule. In one embodiment, the isolated polypeptide molecule comprises a molecule selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO :53, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136 SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, a sequence of a population consisting of SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, and SEQ ID NO: 149, or a sequence having 95% to 99% identity thereto.

In another aspect, the invention relates to a nucleic acid molecule encoding a chimeric antigen receptor (CAR) molecule comprising an anti-BCMA binding domain, a transmembrane domain, and an intracellular signal comprising a stimulatory domain a transduction domain, and wherein the anti-BCMA binding domain comprises selected from the group consisting of SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO: 44. SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO:53, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135,SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, a sequence of a population consisting of SEQ ID NO: 148 and SEQ ID NO: 149, or a sequence having 95% to 99% identity thereto.

In one embodiment, the encoded CAR molecule further comprises a sequence encoding a costimulatory domain. In one embodiment, the costimulatory domain is, for example, a functional signaling domain of a protein selected from the group consisting of: MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, for example, as described herein. , cytokine receptor, integrin, signaling lymphocyte activating molecule (SLAM protein), activated NK cell receptor, BTLA, Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS , ICAM-1, LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7 , NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 ( SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a And a ligand that specifically binds to CD83. In one embodiment, the costimulatory domain comprises the sequence of SEQ ID NO:7. In one embodiment, the transmembrane domain is, for example, as described herein, for example, a transmembrane domain of a protein selected from the group consisting of: alpha, beta or ξ chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154. In one embodiment, the transmembrane domain comprises the sequence of SEQ ID NO: 6. In one embodiment, the intracellular signaling domain comprises a functional signaling domain of 4-1BB and a functional signaling domain of purine. In one embodiment, the intracellular signaling domain comprises the sequence of SEQ ID NO:7 and the sequence of SEQ ID NO:9, wherein the sequences comprising the intracellular signaling domain are represented in the same framework and are a single polypeptide Chain form. In one embodiment, the anti-BCMA binding domain is joined to the transmembrane domain by a hinge region. In one embodiment, the hinge region comprises SEQ ID NO:2. In one embodiment, the hinge region comprises SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5.

In another aspect, the invention relates to an encoded CAR molecule comprising a leader sequence of SEQ ID NO: 1, having a selection selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, a group consisting of SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, and SEQ ID NO: 149 a sequence or a scFv domain thereof having a sequence of 95%-99% identity, a hinge region of SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5, having SEQ ID NO The transmembrane domain of the sequence of 6 and the 4-1BB having the sequence of SEQ ID NO: a CD27 co-stimulatory domain having a stimulatory domain or a CD27 co-stimulatory domain having the sequence of SEQ ID NO: 8 or having a sequence of SEQ ID NO: 1104 (or a sequence having 95%-99% identity thereto) or having SEQ ID NO: 1106 sequence (or has The ICOS co-stimulatory domain of the sequence of 95%-99% identity, and the CD3ξ stimulation domain having the sequence of SEQ ID NO: 9 or SEQ ID NO: 10. In one embodiment, the encoded CAR molecule comprises a strain selected from the group consisting of SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, The sequence of the population consisting of SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, and SEQ ID NO: 233, or a sequence with 95%-99% identity thereto.

The nucleic acid sequence encoding the desired molecule can be obtained using recombinant methods known in the art, such as by screening a library of cells from a gene, using a standard technique, obtaining a gene from a vector known to include the gene, or by self-containing a gene. The cells and tissues are directly separated. Alternatively, related genes can be produced synthetically rather than colonized.

The present invention also provides a vector into which the DNA of the present invention is inserted. Vectors derived from retroviruses (such as lentiviruses) are suitable tools for achieving long-term gene transfer because they allow long-term stable integration of the transgenic genes and their propagation in daughter cells. An additional advantage of lentiviral vectors over vectors derived from oncogenic retroviruses, such as murine leukemia virus, is that they can transduce non-proliferating cells, such as hepatocytes. It also has the added advantage of low immunogenicity. The retroviral vector can also be, for example, a gamma retroviral vector. A gamma retroviral vector can include, for example, a promoter, a packaging signal (Ψ), a primer binding site (PBS), one or more (eg, two) long terminal repeats (LTRs), and related transgenes, such as a coding CAR The gene. The gamma retroviral vector can lack viral structural genes such as gag, pol and env. Exemplary gamma inversion Viral vectors include murine leukemia virus (MLV), splenic foci forming virus (SFFV), and myeloplastic sarcoma virus (MPSV), and vectors derived therefrom. Other gamma retroviral vectors are described, for example, in Tobias Maetzig et al., "Gammaretroviral Vectors: Biology, Technology and Application" Viruses. June 2011; 3(6): 677-713.

In another embodiment, the vector comprising the nucleic acid encoding the desired CAR of the invention is an adenoviral vector (A5/35). In another embodiment, the expression of a nucleic acid encoding a CAR can be achieved using a transposon such as sleeping beauty, CRISPR, CAS9, and zinc finger nuclease. See below June et al, 2009 Nature Reviews Immunology 9.10: 704-716, which is incorporated herein by reference.

Briefly, the expression of a natural or synthetic nucleic acid encoding a CAR is typically achieved by operably linking a nucleic acid encoding a CAR polypeptide or portion thereof to a promoter and incorporating the construct into a performance vector. The vector can be adapted to replicate and integrate eukaryotes. A typical selection vector contains a transcriptional and translational terminator, a starting sequence and a promoter suitable for regulating the expression of the desired nucleic acid sequence.

The expression constructs of the invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Gene delivery methods are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5, 580, 859, 5, 589, 466, incorporated herein by reference. In another embodiment, the invention provides a gene therapy vector.

Nucleic acids are selected for use in many types of vectors. For example, nucleic acids can be selected for inclusion in vectors including, but not limited to, plastids, phagemids, phage derivatives, animal viruses, and vesicles. Carriers of particular interest include expression vectors, replication vectors, probe production vectors, and sequencing vectors.

Furthermore, the expression vector can be provided to the cells in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, Vol. 1 - Volume 4, Cold Spring. Harbor Press, NY), and other virology and molecular virology manuals. Suitable viruses for use include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, suitable vectors contain an origin of replication that functions in at least one organism, a promoter sequence, a suitable restriction endonuclease site, and one or more selectable markers (eg, WO 01/96584; WO 01/ 29058; and U.S. Patent No. 6,326,193).

Many viral-based systems have been developed to transfer genes into mammalian cells. For example, retroviruses provide a suitable platform for gene delivery systems. The selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the individual either in vivo or ex vivo. Many retroviral systems are known in the art. In some embodiments, an adenoviral vector is used. Many adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

Additional promoter elements (eg, enhancers) regulate the frequency of transcription initiation. Typically, such elements are located in the 30-110 bp region upstream of the start site, although many promoters have been shown to also contain functional elements downstream of the start site. The spacing between the promoter elements is typically flexible such that the promoter function is retained when the elements are inverted or moved relative to each other. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decrease. Depending on the promoter, it appears that individual elements can be used cooperatively or independently to activate transcription.

An example of a promoter capable of expressing a CAR transgene in mammalian T cells is the EF1a promoter. The native EF1a promoter drives the expression of the alpha subunit of the elongation factor-1 complex, which is responsible for the delivery of the amine thiol tRNA to the ribosome by the enzyme. The EF1a promoter has been widely used in mammals to express plastids and has been shown to efficiently drive transgenic genes that are self-selected into lentiviral vectors to express CAR. See, for example, Milone et al, Mol. Ther. 17(8): 1453-1464 (2009). In one aspect, the EF1a promoter comprises the sequence provided as SEQ ID NO:11.

Another example of a promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving a high degree of expression of any of the polynucleotide sequences operably linked thereto. However, other constitutive promoter sequences can also be used, including but not limited to prion 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) a promoter, a MoMuLV promoter, an avian leukemia virus promoter, an immediate early promoter of Epstein-Barr virus, a Rous sarcoma virus promoter, and a human gene promoter such as, but not limited to Actin promoter, myosin promoter, elongation factor-1 alpha promoter, hemoglobin promoter and creatine kinase promoter. Furthermore, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter provides a molecular switch that is capable of shutting down performance when such expression is turned on or when performance is not required to be performed when a polynucleotide sequence that is operably linked thereto is required to be expressed. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

Another example of a promoter is the phosphoglycerate kinase (PGK) promoter. In embodiments, a truncated PGK promoter may be required (eg, having one or more, eg, 1, 2, 5, 10, 100, 200, 300 or 400 nucleotides compared to the wild-type PGK promoter sequence) Missing PGK promoter). The nucleotide sequence of an exemplary PGK promoter is provided below.

WT PGK promoter (SEQ ID NO: 1109)

An exemplary truncated PGK promoter: PGK100: ACCCCTCTCTCCAGCCACTAAGCCAGTTGCTCCCTCGGCTGACGGCTGCACGCGAGGCCTCCGAACGTCTTACGCCTTGTGGCGCGCCCGTCCTTGTCCCGGGTGTGATGGCGGGGTG (SEQ ID NO: 1110)

PGK200: (SEQ ID NO: 1111)

PGK300: (SEQ ID NO: 1112)

PGK400: (SEQ ID NO: 1113)

Vectors may also include, for example, a signal sequence that promotes secretion, a polyadenylation signal and a transcription terminator (eg, from a bovine growth hormone (BGH) gene), elements that permit episomal replication, and replication in prokaryotes (eg, SV40 origin and ColE1). Or other elements known in the art) and/or elements that allow for selection (eg, ampicillin resistance gene and/or zeocin marker).

To assess the performance of a CAR polypeptide or portion thereof, the expression vector to be introduced into the cell may also contain a selectable marker gene or reporter gene or both to facilitate identification and selection of the expression cell from a population of cells that are attempting to transfect or infect via the viral vector. In other aspects, the selectable marker can be carried on separate DNA sheets and used in a co-transfection procedure. Both the selectable marker and the reporter gene can be flanked by appropriate regulatory sequences to enable expression in the host cell. Suitable selectable markers include, for example, antibiotic resistant genes such as neo and its analogs.

The reporter gene is used to identify potential transfected cells and to assess the functionality of the regulatory sequences. In general, a reporter gene is a gene that does not or does not exhibit in an organism or tissue and encodes a polypeptide that exhibits a property that is readily detectable (eg, enzymatic activity). The performance of the reporter gene is analyzed at a suitable time after the DNA has been introduced into the recipient cell. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenic acid acetyltransferase, secreted alkaline phosphatase or green fluorescent protein genes (eg Ui-Tei et al., 2000). FEBS Letters 479: 79-82). Suitable performance systems are well known and can be prepared or purchased using known techniques. In general, a construct having a minimal 5' flanking region showing the highest degree of expression of a reporter gene is identified as a promoter. Such a promoter region can be ligated to a reporter gene and used to assess the ability of the agent to modulate the transcription driven by the promoter.

In one embodiment, the vector may further comprise a nucleic acid encoding a second CAR. In one embodiment, the second CAR comprises an antigen binding domain directed against a target expressed on acute myeloid leukemia cells, such as CD123, CD34, CLL-1, folate receptor β or FLT3; Targets expressed on cells, such as the antigen binding domain of CD10, CD19, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b or CD79a. In one embodiment, the vector comprises a nucleic acid sequence encoding a first CAR that specifically binds to the first antigen and includes an intracellular signaling domain having a costimulatory signaling domain but no major signaling domain And a nucleic acid encoding a second CAR that specifically binds to a second different antigen and includes an intracellular signaling domain having a major signaling domain but no co-stimulatory signaling domain. In one embodiment, the vector comprises a nucleic acid encoding a first BCMA CAR comprising a BCMA binding domain, a transmembrane domain and a costimulatory domain; and a nucleic acid encoding a second CAR, the second CAR target An antigen other than BCMA (eg, an antigen expressed on AML cells, such as CD123, CD34, CLL-1, folate receptor β or FLT3; or an antigen expressed on B cells, such as CD10, CD19, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b or CD79a), It also includes an antigen binding domain, a transmembrane domain, and a major signaling domain. In another embodiment, the vector comprises a nucleic acid encoding a first BCMA CAR, the first BCMA CAR comprising a BCMA binding domain, a transmembrane domain and a major signaling domain; and a nucleic acid encoding a second CAR, the second CAR specifically binds antigens other than BCMA (eg, antigens expressed on AML cells, such as CD123, CD34, CLL-1, folate receptor β or FLT3; or antigens expressed on B cells, such as CD10, CD19 , CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b or CD79a) and includes an antigen binding domain, a transmembrane domain and a costimulatory signaling domain for the antigen.

In one embodiment, the vector comprises a nucleic acid encoding a BCMA CAR as described herein and a nucleic acid encoding a CAR inhibiting. In one embodiment, the inhibitory CAR comprises an antigen binding domain that binds to an antigen found on normal cells (eg, normal cells that also exhibit BCMA) rather than cancer cells. In one embodiment, the inhibitory CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular domain of an inhibitory molecule. For example, the intracellular domain that inhibits CAR can be PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (eg, CEACAM-1, CEACAM-3, and/or CEACAM-5), LAG3, VISTA, BTLA , TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine And the intracellular domain of TGFR β.

In embodiments, a vector may comprise two or more nucleic acid sequences encoding a CAR, such as the BCMA CAR described herein, and a second CAR, eg, inhibiting CAR or specifically binding to an antigen other than BCMA (eg, Antigens expressed on AML cells, such as CD123, CLL-1, CD34, FLT3 or folate receptor β; or antigens expressed on B cells, such as CD10, CD19, CD20, CD22, CD34, CD123, FLT-3 CAR of ROR1, CD79b, CD179b or CD79a). In such embodiments, two or more nucleic acid sequences encoding a CAR are in a single frame in the form of a single polypeptide chain. Nucleic acid molecule coding. In this aspect, two or more CARs can be separated, for example, by one or more peptide cleavage sites (eg, an autologous cleavage site or an endogenous protease). Examples of peptide cleavage sites include the following, wherein the GSG residue is optionally present: T2A: (GSG) E G R G S L L T C G D V E E N P G P (SEQ ID NO: 1114)

P2A: (GSG) A T N F S L L K Q A G D V E E N P G P (SEQ ID NO: 1115)

E2A: (GSG) Q C T N Y A L L K L A G D V E S N P G P (SEQ ID NO: 1116)

F2A: (GSG)V K Q T L N F D L L K L A G D V E S N P G P (SEQ ID NO: 1117)

Methods for introducing and expressing genes into cells are known in the art. In the case of a performance vector, the vector is readily introduced into a host cell (e.g., a mammalian, bacterial, yeast or insect cell) by any of the methods of the art. For example, a performance vector can be transferred to a host cell by physical, chemical or biological means.

Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, Vol. 1 - Volume 4, Cold Spring Harbor Press, NY. A preferred method for introducing a polynucleotide into a host cell is calcium phosphate transfection.

Biological methods for introducing related polynucleotides into host cells include the use of DNA and RNA vectors. Viral vectors and especially retroviral vectors have become the most widely used method for inserting genes into mammals, such as human cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus type I, adenoviruses, and adeno-associated viruses and the like. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, These include oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as an in vitro and in vivo delivery vehicle is a liposome (e.g., an artificial blister). Other methods of targeting nucleic acids that are currently advanced in the art are available, such as delivery of polynucleotides using targeted nanoparticle or other suitable submicron size delivery systems.

In the case of utilizing a non-viral delivery system, exemplary delivery vehicles are liposomes. It is contemplated that a lipid formulation can be used to introduce a nucleic acid into a host cell (in vitro, ex vivo or in vivo). In another aspect, the nucleic acid can be associated with a lipid. The nucleic acid associated with the lipid may be encapsulated in the aqueous interior of the liposome, interspersed within the lipid bilayer of the liposome, attached to the liposome via a linker molecule associated with both the liposome and the oligonucleotide, coated In liposome, complexed with liposomes, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, contained in lipids as a suspension, containing microvesicles or complexed with microvesicles, or otherwise associated with lipids Association. The composition associated with the lipid, lipid/DNA or lipid/expression carrier is not limited to any particular structure in solution. For example, it may be present in a bilayer structure, in the form of micelles or having a "collapsed" structure. It can also be simply interspersed into the solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which may be naturally occurring lipids or synthetic lipids. For example, lipids include fat droplets naturally occurring in the cytoplasm and classes of compounds containing long chain aliphatic hydrocarbons and derivatives thereof (such as fatty acids, alcohols, amines, amine alcohols, and aldehydes).

Suitable lipids are available from commercial sources. For example, dimyristoylphosphatidylcholine ("DMPC") is available from Sigma, St. Louis, MO; tridodecyl phosphate ("DCP") is available from K & K Laboratories (Plainview, NY); cholesterol ("Choi") is available from Calbiochem-Behring; dimyristoylphospholipid glycerol ("DMPG") and other lipids are available from Avanti Polar Lipids, Inc. (Birmingham, AL.). A stock solution of the lipid in chloroform or chloroform/methanol can be stored at about -20 °C. Chloroform is used as the sole solvent because it evaporates more easily than methanol. "Liposomes" are general techniques encompassing a variety of monolayer and multilamellar lipid mediators formed by the production of blocked lipid bilayers or aggregates. language. Liposomes are characterized by having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by an aqueous medium. It spontaneously forms when the phospholipid is suspended in an excess of aqueous solution. The lipid component undergoes self-rearrangement prior to forming the closed structure and captures water and dissolved lysate between the lipid bilayers (Ghosh et al, 1991 Glycobiology 5: 505-10). However, compositions having a structure different from normal vesicle structures in solution are also contemplated. For example, the lipid may be present in a micellar structure or only as a non-uniform aggregate of lipid molecules. Lipid amine-nucleic acid complexes are also contemplated.

Regardless of the method for introducing an exogenous nucleic acid into a host cell or otherwise exposing the cell to an inhibitor of the present invention, various assays can be performed to confirm the presence of the recombinant DNA sequence in the host cell. Such assays include, for example, "molecular biology" analysis well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR, and PCR; "biochemical" assays, such as detecting the presence or absence of a particular peptide, Analytical methods for identifying agents within the scope of the invention, for example, by immunological means (ELISA and Western blotting) or by the methods described herein.

The invention further provides a vector comprising a nucleic acid molecule encoding a CAR. In one aspect, the CAR vector can be directly transduced into a cell, such as a T cell or an NK cell. In one aspect, the vector is a selection or expression vector, for example, including but not limited to, one or more vectors (eg, a plastid, a selection vector, a microcircle, a microcarrier, a double minichromosome) , retroviruses and lentiviral vector constructs. In one aspect, the vector is capable of expressing a CAR construct in a mammalian T cell or NK cell. In one aspect, the mammalian T cell is a human T cell or an NK cell. In one aspect, the mammalian NK cells are human NK cells.

Cell source

Prior to amplification and genetic modification, the source of cells, such as immune effector cells (eg, T cells or NK cells), is obtained from the individual. The term "individual" is intended to include living organisms (e.g., mammals) that cause an immune response. Examples of individuals include humans, dogs, cats, mice, rats And its genetically transmitted species. T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissues from infected sites, ascites, pleural effusion, spleen tissue, and tumors.

In certain aspects of the invention, a number of immune effector cells (e.g., T cells or NK cells) available in the art can be used. In certain aspects of the invention, T cells can be obtained from blood units collected from an individual using a number of techniques known to those skilled in the art, such as Ficoll ^(TM) separation. In a preferred aspect, cells from the circulating blood of the individual are obtained by debridement. The scavenging products usually contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one aspect, cells harvested by debridement can be washed to remove the plasma fraction and placed in an appropriate buffer or medium for subsequent processing steps. In one aspect of the invention, the cells are washed with phosphate buffered saline (PBS). In an alternate aspect, the wash solution lacks calcium and may be deficient in magnesium or may lack many, if not all, divalent cations.

The initial activation step in the absence of calcium can result in activation of amplification. As will be readily appreciated by those of ordinary skill in the art, the washing step can be accomplished by methods known to those skilled in the art, such as by using semi-automatic "circulating" centrifuges (e.g., Cobe 2991 cell processor, Baxter CytoMate or Haemonetics cells) according to the manufacturer's instructions. Saver 5). After washing, the cells can be resuspended in various biocompatible buffers, such as Ca-free, Mg-free PBS, PlasmaLyte A, or other physiological saline solution with or without buffer. Alternatively, the undesired components of the debridement sample can be removed and the cells resuspended directly in the culture medium.

It is recognized that the methods of the present application may utilize medium conditions comprising 5% or less, such as 2% human AB serum, and using known media conditions and compositions, such as those described in the following: Smith et al, "Ex vivo expansion of human T cells for adoptive immunotherapy using the novel Xeno-free CTS Immune Cell Serum Replacement" Clinical & Translational Immunology (2015) 4, e31; doi: 10.1038/cti.2014.31.

In one aspect, T cells are isolated from peripheral blood lymphocytes by dissolving red blood cells and depleting the monocytes, for example, by PERCOLLTM gradient centrifugation or by countercurrent centrifugation. Specific subpopulations of T cells (such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells) can be further isolated by positive selection or negative selection techniques. For example, in one aspect, breeding with an anti-CD3/anti-CD28 (eg, 3x28)-bound bead (such as DYNABEADS® M-450 CD3/CD28 T) is sufficient to positively select the desired T cell. The time period is used to separate T cells. In one aspect, the period of time is about 30 minutes. In another aspect, the time period is in the range of 30 minutes to 36 hours or longer and all integer values therebetween. In another aspect, the period of time is at least 1, 2, 3, 4, 5 or 6 hours. In another preferred aspect, the period of time is from 10 to 24 hours. In one aspect, the incubation period is 24 hours. T cells can be isolated using longer incubation times in any situation where there are fewer T cells compared to other cell types, such as tumor infiltrating lymphocytes (TIL) isolated from tumor tissue or immunocompromised individuals. In addition, the use of longer incubation times increases the efficiency of capturing CD8+ T cells. Thus, by simply shortening or prolonging the time, T cells are bound to CD3/CD28 beads, and/or by increasing or decreasing the ratio of beads to T cells (as further described herein), at the beginning of the culture or A subset of T cells is preferred at other time points during the method. In addition, T cell subsets can be preferentially selected at the beginning of the culture or at other desired time points by increasing or decreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on the beads or other surface. Those skilled in the art will appreciate that multiple rounds of selection may also be used in the context of the present invention. In some aspects, a selection procedure may be required and "unselected" cells are used during activation and amplification. "Unselected" cells can also be selected for other rounds.

Concentration of T cell populations by negative selection can be achieved using a combination of antibodies against surface markers unique to negative selection cells. One method is to sort and/or select cells via negative magnetic immunoadhesion or flow cytometry, the use of which is present on negative selection cells A monoclonal antibody mixture of cell surface markers. For example, to attenuate CD4+ cells by negative selection, monoclonal antibody mixtures typically include antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some aspects, it may be desirable to thicken or positively select regulatory T cells that normally express CD4+, CD25+, CD62Lhi, GITR+, and FoxP3+. Alternatively, in certain aspects, T-regulated cells are depleted by anti-C25-bound beads or other similar selection methods.

The methods described herein can include, for example, selection of immune effector cells, such as T cells, as a specific subset of T-regulated cell depletion populations, CD25+ depleted cells, using, for example, negative selection techniques as described herein. Preferably, the T-regulated depleted cell population contains less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% of CD25+ cells.

In one embodiment, T regulatory cells, such as CD25+ T cells, are removed from the population using an anti-CD25 antibody or fragment thereof or a CD25 binding ligand IL-2. In one embodiment, the anti-CD25 antibody or fragment thereof or CD25 binding ligand binds to a substrate such as a bead, or is additionally coated on a substrate such as a bead. In one embodiment, the anti-CD25 antibody or fragment thereof binds to a substrate as described herein.

In one embodiment, a depletion of CD25 from Miltenyi ^TM Reagent T regulatory cells removed from the population, for example, CD25 + T cells. In one embodiment, the ratio of cells to CD25 depleted reagent is 1e7 cells with 20 μL, or 1e7 cells with 15 μL, or 1e7 cells with 10 μL, or 1e7 cells with 5 μL, or 1e7 cells with 2.5 μL, Or 1e7 cells with 1.25 μL. In one embodiment, for example, for T regulatory cells, such as CD25+ depletion, more than 500 million cells/ml are used. In another aspect, a cell concentration of 6, 7, 8, or 900 million cells/ml is used.

In one embodiment, the population of immune effector cells to be depleted comprises about 6 x ¹⁰⁹ CD25+ T cells. In other aspects, be a depletion of the population of immune effector cells comprising from about 1 × 10 ⁹ to 1 × 10 ¹⁰ th any integral value between CD25 + T cells and both. In one embodiment, the resulting population T regulates depleted cells with 2 x ¹⁰⁹ T regulatory cells, such as CD25+ cells, or lower (eg, 1 x 10 ⁹ , 5 x 10 ⁸ , 1 x 10 ⁸ , 5 x 10) ⁷ , 1 × 10 ⁷ or less CD25+ cells).

In one embodiment, T regulatory cells, such as CD25+ cells, are removed from the population using a CliniMAC system with a depletion tube set, such as tube 162-01. In one embodiment, the CliniMAC system operates on a depleted setting such as DEPLETION 2.1.

Without wishing to be bound by a particular theory, reducing the level of negative regulatory factors of immune cells (eg, reducing the number of unwanted immune cells, such as T _REG cells) during an individual's pre-surgery or during the manufacture of a cell product that exhibits CAR may reduce individual recurrence. risk. For example, methods of depleting T _REG cells are known in the art. Methods of reducing T _REG cells include, but are not limited to, cyclophosphamide, anti-GITR antibodies (anti-GITR antibodies described herein), CD25 depletion, and combinations thereof.

In some embodiments, the method of manufacture comprises reducing the number of T _REG cells (eg, depletion) prior to making the cells expressing CAR. For example, a method of manufacture comprises contacting a sample (eg, a scavenging sample) with an anti-GITR antibody and/or an anti-CD25 antibody (or a fragment thereof or a CD25 binding ligand), eg, in the manufacture of a cell that exhibits CAR (eg, T cells, NK cells) pre-depleted T _REG cells.

In one embodiment, the individual is pretreated with one or more therapies that reduce T _REG cells prior to collecting the cells for use in making the cell product indicative of CAR, thereby reducing the risk of the individual facing a relapse of the cell treatment for CAR. In one embodiment, the method of reducing T _REG cells includes, but is not limited to, administering to the individual one or more of cyclophosphamide, anti-GITR antibody, CD25 depletion, or a combination thereof. Administration to one or more of cyclophosphamide, anti-GITR antibody, CD25 depletion, or a combination thereof can be performed before, during or after infusion of a cell product expressing CAR.

In one embodiment, the individual is pretreated with cyclophosphamide prior to collecting the cells for use in the production of a cell product that expresses CAR, thereby reducing the risk of the individual facing a relapse of the cell treatment of the CAR. In one embodiment, the cells are collected for use in the manufacture of a cell product that exhibits CAR The individual is pre-treated with an anti-GITR antibody, thereby reducing the risk of the individual facing relapse of the cell treated with CAR.

In one embodiment, the population of cells to be removed is neither a regulatory T cell nor a tumor cell, but is a cell that otherwise negatively affects the expansion and/or function of the CART cell, eg, exhibits CD14, CD11b, CD33, CD15 Or cells that are expressed by other markers of potential immunosuppressive cells. In one embodiment, such cells are envisioned to be removed in parallel with regulatory T cells and/or tumor cells, or removed after the depletion, or removed in another order.

The methods described herein can include more than one selection step, such as more than one depletion step. Concentration of a population of T cells by negative selection can be achieved, for example, using a combination of antibodies against surface markers unique to negative selection cells. One method is to sort and/or select cells via negative magnetic immunoadhesion or flow cytometry using a mixture of monoclonal antibodies directed against cell surface markers present on negative selection cells. For example, to attenuate CD4+ cells by negative selection, monoclonal antibody mixtures can include antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8.

The methods described herein can further comprise removing cells from a population exhibiting a tumor antigen, such as a tumor antigen that does not comprise CD25, such as CD19, CD30, CD38, CD123, CD20, CD14 or CD11b, thereby providing a suitable CAR for expression, for example The T regulation of CAR as described herein is depleted, such as CD25+ depletion and tumor antigen depleted cell populations. In one embodiment, the cells expressing the tumor antigen are removed simultaneously with T regulation, such as CD25+ cells. For example, an anti-CD25 antibody or fragment thereof and an anti-tumor antigen antibody or fragment thereof can be attached to the same substrate that can be used to remove cells, such as beads, or an anti-CD25 antibody or fragment thereof or an anti-tumor antigen antibody or Fragments thereof can be attached to separate beads that the mixture can be used to remove cells. In other embodiments, the removal of T regulatory cells, such as CD25+ cells, and the removal of tumor antigen expressing cells are continuous and can be performed, for example, in either order.

Also provided are cells comprising a population removal performance checkpoint inhibitor (such as a checkpoint inhibitor as described herein), such as one of PD1+ cells, LAG3+ cells, and TIM3+ cells. Or a plurality of methods whereby a T-regulated depletion, such as a CD25+ depleted cell and a checkpoint inhibitor depleted cell, such as a population of PDl+, LAG3+ and/or TIM3+ depleted cells, is provided. Exemplary checkpoint inhibitors include PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (eg CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine and TGFR beta. In an embodiment, the checkpoint inhibitor is PD1 or PD-L1. In one embodiment, cells exhibiting a checkpoint inhibitor are removed simultaneously with Tregulation, such as CD25+ cells. For example, an anti-CD25 antibody or fragment thereof and an anti-spot inhibitor antibody or fragment thereof can be attached to the same bead that can be used to remove cells, or an anti-CD25 antibody or fragment thereof or an anti-tumor antigen antibody or Fragments thereof can be attached to separate beads that the mixture can be used to remove cells. In other embodiments, the removal of T regulatory cells, such as CD25+ cells, and the removal of checkpoint inhibitors, the cells are continuous and can be performed, for example, in either order.

In one embodiment, a population of T cells expressing one or more of the following may be selected: IFN-γ, TNFα, IL-17A, IL-2, IL-3, IL-4, GM-CSF, IL-10 , IL-13, granzyme B and perforin, or other suitable molecule, such as other cytokines. A method of screening for cell expression can be determined, for example, by the method described in PCT Publication No. WO 2013/126712.

To separate a desired population of cells by positive or negative selection, cell concentration and surface (eg, particles, such as beads) can vary. In some aspects, it may be desirable to significantly reduce the volume of beads and cells mixed together (e.g., increase cell concentration) to ensure that cells are in maximum contact with the beads. For example, in one aspect, a concentration of 2000,000,000 cells/ml is used. In one aspect, a concentration of 1,000,000,000 cells/ml is used. In another aspect, greater than 100,000,000 cells/ml are used. In another aspect, 10,000,000, 15,000,000, 20,000,000, 25,000,000, 30,000,000, 35,000,000, 40,000,000, 45,000,000 or 50,000,000 cells/ml. In another aspect, a concentration of 75,000,000, 80,000,000, 85,000,000, 90,000,000, 95,000,000 or 100,000,000 cells/ml is used. In other aspects, a concentration of 125,000,000 or 150,000,000 cells/ml can be used. Use of high concentrations can result in increased cell yield, cell activation, and cell expansion. In addition, the use of high cell concentrations allows for more efficient capture of cells that may be weakly associated with a target antigen (such as CD28-negative T cells) or from cells in which many tumor cells are present (eg, leukemia blood, tumor tissue, etc.) to more efficiently capture cells. Such cell populations can have therapeutic value and will need to be obtained. For example, the use of high concentration cells allows for more efficient selection of CD8+ T cells that typically have weaker CD28 expression.

In related aspects, it may be desirable to use cells at lower concentrations. The interaction between the particles and the cells is minimized by significantly diluting the mixture of T cells and surfaces such as particles, such as beads. This selection represents a large number of cells to be bound to the desired antigen of the particle. For example, CD4+ T cells exhibit a higher level of CD28 and are more efficiently captured than CD8+ T cells at dilute concentrations. In one aspect, the cells used in a concentration of 5 × 10 ⁶ / ml. In other aspects, the concentration used may range from about 1 x 10 ⁵ /ml to 1 x 10 ⁶ /ml, and any integer value therebetween.

In other aspects, the cells can be incubated on a rotator at 2-10 ° C or room temperature for varying lengths of time at varying speeds.

T cells for stimulation can also be frozen after the washing step. Without wishing to be bound by theory, the freezing and subsequent thawing steps provide a more uniform product by removing granulocytes from the cell population and removing monocytes to some extent. After the washing step of removing plasma and platelets, the cells can be suspended in the frozen solution. Although many frozen solutions and parameters are known in the art and will be suitable for this situation, one method involves the use of PBS containing 20% DMSO and 8% human serum albumin, or containing 10% polydextrose 40 and 5% right. Rotose, 20% human serum albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% right-handed a medium of sugar 5%, 0.45% NaCl, 10% polydextrose 40 and 5% dextrose, 20% human serum albumin and 7.5% DMSO or other suitable cell freezing medium containing, for example, Hespan and PlasmaLyte A, cells then It was chilled to -80 ° C at a rate of 1 ° per minute and stored in the gas phase of a liquid nitrogen storage tank. Other methods of controlled freezing and uncontrolled freezing at -20 ° C or in liquid nitrogen can be used.

In some aspects, cryopreserved cells are thawed and washed as described herein and allowed to stand at room temperature for one hour, followed by activation using the methods of the invention.

It is also contemplated in the context of the present invention to collect a blood sample or a clearance product from an individual for a period of time prior to the need to amplify the cells as described herein. Thus, the source of cells to be expanded can be collected at any desired point in time, and the desired cells (such as immune effector cells, such as T cells or NK cells) can be isolated and frozen for subsequent use in a number of ways to benefit from cell therapies, such as T cells. Cell therapy for a disease or condition of a therapy, such as a disease or condition as described herein, such as in T cell therapy. In one aspect, the blood sample or removal is taken from a generally healthy individual. In some aspects, the blood sample or removal is taken from a generally healthy individual at risk of developing the disease but no disease has occurred, and the relevant cells are isolated and frozen for subsequent use. In some aspects, immune effector cells (eg, T cells or NK cells) can be expanded, frozen, and used later. In some aspects, samples are collected from a patient shortly after diagnosis of a particular disease as described herein but prior to any treatment. In another aspect, the cells are isolated from individual blood samples or clearance prior to a number of related treatment modalities including, but not limited to, the use of, for example, natalizumab, efavizumab (efalizumab), antiviral agents, chemotherapy, radiation, immunosuppressive agents (such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506) , antibodies or other immunosuppressive agents (such as CAMPATH, anti-CD3 antibodies, cels cedar, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228) Drug and irradiation treatment.

In another aspect of the invention, the T cells are obtained directly from the patient following treatment with the individual together with the functional T cells. In this regard, it has been observed that after treatment with certain cancers, especially after treatment with drugs that destroy the immune system, the ability of the obtained T cell quality for its ex vivo expansion is shortly after the treatment of the patient usually recovers from the treatment. It can be best or improved. Likewise, after ex vivo manipulation using the methods described herein, such cells may be in a preferred state to enhance transplantation and in vivo expansion. Therefore, collection of blood cells (including T cells, dendritic cells, or other cells of the hematopoietic lineage) at this stage of recovery is encompassed by the present invention. In addition, in some aspects, movement (eg, with GM-CSF movement) and adjustment protocols can be used to establish conditions within the individual, wherein the defined time window, particularly after treatment, should be reproliferated, recirculated, regenerated, and/or Amplify specific cell types. Illustrative cell types include T cells, B cells, dendritic cells, and other cells of the immune system.

In one embodiment, an immune effector cell that expresses a CAR molecule, such as a CAR molecule described herein, is obtained from an individual receiving a low immunopotentiating dose of an mTOR inhibitor. In one embodiment, the population of immune effector cells, such as T cells, to be engineered to express CAR is harvested after a sufficient time to administer a low immunopotentiating dose of the mTOR inhibitor or after a sufficient dose is administered, such that the individual is The ratio of PD1 negative immune effector cells (eg, T cells) or PD1 negative immune effector cells (eg, T cells) / PD1 positive immune effector cells (eg, T cells) harvested from an individual is at least transiently increased.

In other embodiments, a population of immune effector cells, such as T cells, that have been engineered to express CAR can increase the number of PD1 negative immune effector cells (eg, T cells) or increase PD1 negative immune effector cells by contact (eg, An amount of mTOR inhibitor in a ratio of T cells) / PD1 positive immune effector cells (eg, T cells) is treated ex vivo.

In one embodiment, the T cell population lacks diacylglycerol kinase (DGK). DGK deficient cells include cells that do not exhibit DGK RNA or protein or have reduced or inhibited DGK activity. DGK-deficient cells can be produced by genetic methods, such as administration of RNA interference agents (eg, siRNA, shRNA, miRNA) to reduce or prevent DGK expression. Or, DGK Lack of cells can be produced by treatment with a DGK inhibitor as described herein.

In one embodiment, the T cell population lacks Ikaros. Ikaros-deficient cells include cells that do not exhibit Ikaros RNA or protein, or have reduced or inhibited Ikaros activity, and Ikaros-deficient cells can be produced by genetic methods, such as administration of RNA interference agents (eg, siRNA, shRNA, miRNA) to reduce or prevent Ikaros performance. Alternatively, Ikaros-deficient cells can be produced by treatment with an Ikaros inhibitor such as lenalidomide.

In the examples, the T cell population lacks DGK and lacks Ikaros, for example, does not exhibit DGK and Ikaros, or has reduced or inhibited DGK and Ikaros activity. Such DGK and Ikaros deficient cells can be produced by any of the methods described herein.

In one embodiment, the NK cells are obtained from an individual. In another embodiment, the NK cell is an NK cell line, such as the NK-92 cell line (Conkwest).

Allogeneic CAR

In the embodiments described herein, the immune effector cells can be allogeneic immune effector cells, such as T cells or NK cells. For example, the cells can be allogeneic T cells, such as allogeneic T cells that lack functional T cell receptor (TCR) and/or human leukocyte antigen (HLA) (eg, HLA class I and/or HLA class II). .

A T cell lacking a functional TCR can, for example, be engineered such that it does not exhibit any functional TCR on its surface, engineered such that it does not exhibit one or more subunits that constitute a functional TCR (eg, engineered such that it does not exhibit TCR alpha, TCR beta, TCR gamma, TCR δ, TCR ε and/or TCR ζ (or reduced display) are either engineered to produce minimal functional TCR on their surface. Alternatively, a T cell can exhibit a substantially impaired TCR, such as one or more TCR subunits that exhibit a mutant or truncated form. The term "substantially damaged TCR" means that this TCR will not cause an adverse immune response in the host.

The T cells described herein can, for example, be engineered such that they do not exhibit functional HLA on their surface. For example, the T cells described herein can be engineered to make HLA (eg The cell surface appearance of HLA class I and/or HLA class II is down-regulated. In some aspects, HLA down-regulation can be achieved by reducing or eliminating the expression of beta-2 microglobulin (B2M). In some embodiments, T cells may lack functional TCRs and functional HLAs, such as HLA class I and/or HLA class II.

Modified T cells that lack functional TCR and/or HLA expression can be obtained by any suitable means, including gene knocking or blocking gene expression of one or more TCR or HLA subunits. For example, T cells can include blocking TCR using siRNA, shRNA, clustered regular epoch repeats (CRISPR), transcriptional activator-like effector nuclease (TALEN), or zinc finger endonuclease (ZFN). / or HLA gene performance.

In some embodiments, the allogeneic cells can be cells that do not exhibit or exhibit a low degree of inhibitory molecules, such as cells engineered by any of the methods described herein. For example, the cells may be cells that exhibit no or a low degree of expression of an inhibitory molecule, for example, which reduces the ability of a cell expressing CAR to establish an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (eg, CEACAM-1, CEACAM-3, and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine and TGFR beta. Inhibition of inhibitory molecules, such as inhibition at the DNA, RNA or protein level, optimizes cell performance in expressing CAR. In embodiments, for example, an inhibitory nucleic acid, such as an inhibitory nucleic acid, such as a dsRNA, such as siRNA or shRNA, a cluster of regularly spaced short palindromic repeats (CRISPR), a transcriptional activator-like effector nuclease (TALEN) can be used as described herein. Or zinc finger endonuclease (ZFN).

siRNA and shRNA that inhibit TCR or HLA

In some embodiments, targeted cells can be used, such as TCRs and/or HLAs encoded in T cells, and/or inhibitory molecules described herein (eg, PD1, PD-L1, PD-L2) CTLA4, TIM3, CEACAM (eg CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 siRNA or shRNA of a nucleic acid of (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine and TGFR β) inhibits TCR expression and/or HLA expression.

The performance of siRNA and shRNAs in T cells can be achieved using any conventional expression system, such as a lentiviral expression system.

Exemplary shRNAs that downregulate the performance of the components of the TCR are described, for example, in U.S. Publication No. 2012/0321667. Exemplary siRNAs and shRNAs that down-regulate HLA class I and/or HLA class II gene expression are described, for example, in US Publication No. US 2007/0036773.

CRS that inhibits TCR or HLA

As used herein, "CRISPR" or "CRISR for TCR and/or HLA" or "CRISPR for inhibiting TCR and/or HLA" refers to a set of short palindrome repeats of cluster intervals or systems containing such sets of repeats. As used herein, "Cas" refers to a CRISPR-related protein. "CRISPR/Cas" system refers to a system derived from CRISPR and Cas that can be used to silence or mutate TCR and/or HLA genes, and/or inhibitory molecules described herein (eg, PD1, PD-L1, PD-L2) CTLA4, TIM3, CEACAM (eg CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine and TGFR β).

The naturally occurring CRISPR/Cas system is found in approximately 40% of the sequenced eubacterial genome and 90% of the sequenced archaea. Grissa et al. (2007) BMC Bioinformatics 8:172. This system is a type of prokaryotic immune system that confers resistance to foreign genetic elements, such as plastids and phage, and provides an acquired immunological form. Barrangou et al, (2007) Science 315: 1709-1712; Marragini et al, (2008) Science 322: 1843-1845.

The CRISPR/Cas system has been modified for gene editing (silencing, augmenting or altering specific genes) in eukaryotes such as mice or primates. Wiedenheft et al. (2012) Nature 482:331-8. This is achieved by introducing into the eukaryotic cell a plastid containing a specific design of CRISPR and one or more appropriate Cas.

The CRISPR sequence is sometimes referred to as the CRISPR locus and contains alternating repeats and spacers. In naturally occurring CRISPR, the spacer typically comprises a bacterial exogenous sequence, such as a plastid or phage sequence; in the TCR and/or HLA CRISPR/Cas system, the spacer is derived from a TCR or HLA gene sequence.

RNA from the CRISPR locus is constitutively expressed and processed into small RNA by Cas protein. These include spacers that flank the repeats. RNA directs other Cas proteins to silence exogenous genetic elements at RNA or DNA levels. Horvath et al, (2010) Science 327: 167-170; Makarova et al, (2006) Biology Direct 1:7. The spacer thus serves as a template for the RNA molecule, similar to siRNA. Pennisi (2013) Science 341:833-836.

Because these naturally exist in many different types of bacteria, the precise alignment of CRISPR and the structure, function, and number of Cas genes, as well as their products, vary slightly from species to species. Haft et al. (2005) PLoS Comput. Biol. 1: e60; Kunin et al., (2007) Genome Biol. 8: R61; Mojica et al., (2005) J. Mol. Evol. 60: 174-182; Et al., (2005) Microbiol. 151:2551-2561; Pourcel et al., (2005) Microbiol. 151:653-663; and Stern et al., (2010) Trends. Genet. 28: 335-340 . For example, a Cse (Cas subtype, E. coli) protein (eg, CasA) forms a functional complex, Cascade, which processes the CRISPR RNA transcript into a spacer-repeat unit that retains Cascade. Brouns et al. (2008) Science 321:960-964. In other prokaryotes, Cas6 processes CRISPR transcripts. CRISPR-based phage inactivation in E. coli requires Cascade and Cas3, not Cas1 or Cas2. The Cmr (Cas RAMP module) protein in Pyrococcus furiosus and other prokaryotes forms a functional complex with small CRISPR RNA that recognizes and cleaves the complementary target RNA. The simpler CRISPR system relies on the protein Cas9, which is a nuclease with two active cleavage sites, one for each strand of the double helix. Cas9 can be used in gene editing systems in combination with modified CRISPR locus RNA. Pennisi (2013) Science 341:833-836.

The CRISPR/Cas system can therefore be used to edit TCR and/or HLA genes (add or delete base pairs), or introduce pre-terminators, thus reducing TCR and/or HLA performance. Alternatively, the CRISPR/Cas system can be used to reverse the TCR and/or HLA genes in a reversible manner, as in the case of RNA interference. In mammalian cells, for example, RNA can direct the Cas protein to the TCR and/or HLA promoter, spatially blocking RNA polymerase.

Artificial CRISPR/Cas systems that inhibit TCR and/or HLA can be produced using techniques known in the art, such as those described in U.S. Publication No. 20140068797 and Cong (2013) Science 339:819-823. Other artificial CRISPR/Cas systems known in the art to inhibit TCR and/or HLA can also be produced, for example, Tsai (2014) Nature Biotechnol. , 32:6 569-576, U.S. Patent No. 8,871,445; No. 8,865,406; 8,795,965; 8,771,945 and 8,697,359.

TALEN inhibiting TCR and/or HLA

"TALEN" or "TALEN for HLA and/or TCR" or "TALEN for inhibition of HLA and/or TCR" refers to transcriptional activator-like effect nucleases, one that can be used to edit HLA and/or TCR genes, and/or The inhibitory molecule (eg, PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (eg CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160 Labor of 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine and TGFR β) Nuclease.

TALEN is artificially produced by fusing the TAL effector DNA binding domain to a DNA cleavage domain. Transcriptional activator-like effects (TALE) can be engineered to bind to any desired DNA sequence, including a portion of the HLA or TCR genes. By combining engineered TALE with a DNA cleavage domain, a restriction enzyme specific for any desired DNA sequence, including HLA or TCR sequences, can be generated. These can then be introduced into cells where they can be used for genome editing. Boch (2011) Nature Biotech. 29: 135-6; and Boch et al, (2009) Science 326: 1509-12; Moscou et al, (2009) Science 326: 3501.

TALE is a protein secreted by Xanthomonas. The DNA binding domain contains highly conserved repeats of 33-34 amino acid sequences, with the exception of the 12th and 13th amino acids. These two positions are highly variable, showing a strong correlation with specific nucleotide recognition. It can therefore be engineered to bind to the desired DNA sequence.

To generate a TALEN, the TALE protein is fused to a nuclease (N) which is a wild-type or mutant FokI endonuclease. Several mutations have been made to FokI for use in TALEN; such as, for example, increased cleavage specificity or activity. Cermak et al., (2011) Nucl. Acids Res. 39: e82; Miller et al., (2011) Nature Biotech. 29: 143-8; Hockemeyer et al., (2011) Nature Biotech. 29: 731-734; Wood et al. (2011) Science 333:307; Doyon et al., (2010) Nature Methods 8: 74-79; Szczepek et al., (2007) Nature Biotech. 25:786-793; and Guo et al., (2010) J .Mol.Biol. 200:96.

The FokI domain acts as a dimer and requires two constructs with unique DNA binding domains in the target genome with appropriate orientation and spacing. The number of amino acid residues between the TALE DNA binding domain and the FokI cleavage domain and the number of bases between two individual TALEN binding sites appear to be important parameters for achieving high activity levels. Miller et al. (2011) Nature Biotech. 29: 143-8.

Double strand breaks (DSB) can be generated using HLA or TCR TALEN inside the cell. If the repair mechanism improperly repairs the break via non-homologous end joining, a mutation can be introduced at the break. For example, improper repair can introduce frame shift mutations. Alternatively, foreign DNA can be introduced into the cell along with TALEN; depending on the foreign DNA sequence and the chromosomal sequence, this The procedure can be used to correct for defects in the HLA or TCR genes or to introduce such defects into the wt HLA or TCR genes, thus reducing HLA or TCR performance.

TALENs specific for sequences in HLA or TCR can be constructed using any of the methods known in the art, including a variety of protocols using modular components. Zhang et al., (2011) Nature Biotech 29: 149-53; Geibler et al., (2011) PLoS ONE 6: . E19509.

Zinc finger nuclease that inhibits HLA and/or TCR

"ZFN" or "zinc finger nuclease" or "ZFN for HLA and/or TCR" or "ZFN for inhibiting HLA and/or TCR" means zinc finger nuclease, one of which can be used to edit HLA and/or TCR genes, And/or inhibitory molecules described herein (eg, PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (eg, CEACAM-1, CEACAM-3, and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT) , LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine and TGFR Artificial nuclease of β).

Like TALEN, ZFN comprises a FokI nuclease domain (or a derivative thereof) fused to a DNA binding domain. In the case of ZFN, the DNA binding domain comprises one or more zinc fingers. Carroll et al, (2011) Genetics Society of America 188: 773-782; and Kim et al, (1996) Proc. Natl. Acad. Sci. USA 93: 1156-1160.

Zinc refers to a small protein structural motif that is stabilized by one or more zinc ions. The zinc finger can comprise, for example, Cys2His2 and can recognize an approximately 3-bp sequence. Multiple zinc fingers of known specificity can be combined to produce a multi-finger polypeptide that recognizes a sequence of about 6, 9, 12, 15 or 18-bp. Multiple selection and modular assembly techniques are zinc fingers (and combinations thereof) for generating recognition specific sequences (including phage display), yeast one-hybrid systems, bacterial one-hybrid and two-hybrid systems, and mammalian cells.

Like TALEN, ZFN must be dimerized to cleave DNA. Therefore, a pair of ZFNs is required to target non-palindromic DNA sites. The two individual ZFNs must bind to the opposite strand of DNA and properly separate their nucleases. Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA 95: 10570-5.

Also like TALEN, ZFN can form a double-strand break in DNA, which can form a frame shift mutation when not properly repaired, resulting in a decrease in the expression and amount of HLA and/or TCR in the cell. ZFN can also be used in conjunction with homologous recombination for mutations in HLA or TCR genes.

ZFN specific for sequences in HLA and/or TCR can be constructed using any of the methods known in the art. See, for example, Provasi (2011) Nature Med. 18: 807-815; Torikai (2013) Blood 122: 1341-1349; Cathomen et al., (2008) Mol. Ther . 16: 1200-7; Guod et al., (2010) J. Mol. Biol. 400:96; and U.S. Patent Publication No. 2011/0158957; U.S. Patent Publication No. 2012/0060230.

Telomerase expression

While not wishing to be bound by any particular theory, in some embodiments, due to telomere shortening in T cells, therapeutic T cells have short-term persistence in patients; therefore, telomerase gene transfection can be extended Telomere of T cells and increase the persistence of T cells in patients. See Carl June, "Adoptive T cell therapy for cancer in the clinic", Journal of Clinical Investigation, 117: 1466-1476 (2007). Thus, in one embodiment, an immune effector cell, such as a T cell, ectopically expresses a telomerase subunit, such as a catalytic subunit of telomerase, such as a TERT, such as hTERT. In some aspects, the invention provides a method of producing a cell that exhibits CAR comprising contacting a cell with a nucleic acid encoding a telomerase subunit, such as a catalytic subunit of telomerase, such as a TERT, such as hTERT. The cells can be contacted with, prior to, or after the nucleic acid is contacted with the construct encoding the CAR.

In one aspect, the invention provides a method of making a population of immune effector cells (eg, T cells, NK cells). In one embodiment, the method comprises: providing a population of immune effector cells (eg, T cells or NK cells), the population of the immune effector cells and the encoding CAR The nucleic acid is contacted; and the immune effector cell population is contacted with a nucleic acid encoding a telomerase subunit, such as hTERT, under conditions that permit CAR and telomerase expression.

In one embodiment, the nucleic acid encoding the telomerase subunit is DNA. In one embodiment, the nucleic acid encoding a telomerase subunit comprises a promoter capable of driving the expression of a telomerase subunit.

In one embodiment, hTERT has the amino acid sequence of GenBank Protein ID AAC51724.1 (Meyerson et al., "hEST2, the Putative Human Telomerase Catalytic Subunit Gene, Is Up-Regulated in Tumor Cells and during Immortalization" Cell No. 90 Vol. 4, August 22, 1997, pp. 785-795): (SEQ ID NO: 284)

In one embodiment, the hTERT has a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:284. In one embodiment, hTERT has the sequence of SEQ ID NO:284. In one embodiment, the hTERT comprises a deletion (eg, no more than 5, 10, 15, 20, or 30 amino acids) at the N-terminus, C-terminus, or both ends. In one embodiment, the hTERT comprises a transgenic amino acid sequence (eg, no more than 5, 10, 15, 20, or 30 amino acids) at the N-terminus, C-terminus, or both ends.

In one embodiment, hTERT is encoded by the nucleic acid sequence of Genbank Accession No. AF018167 (Meyerson et al., "hEST2, the Putative Human Telomerase Catalytic Subunit Gene, Is Up-Regulated in Tumor Cells and during Immortalization" Cell Vol. 90, No. Issue 4, August 22, 1997, pp. 785-795): (SEQ ID NO: 285)

In one embodiment, the hTERT is encoded by a nucleic acid having a sequence that is at least 80%, 85%, 90%, 95%, 96, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:285. In one embodiment, hTERT is encoded by the nucleic acid of SEQ ID NO:285.

Activation and expansion of T cells

T cells can generally be activated and expanded using methods as described in, for example, U.S. Patent Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication No. 20060121005.

In general, T cells of the invention can be amplified by contact with a surface to which a signal that stimulates a CD3/TCR complex is attached and a surface that stimulates a co-stimulatory molecule on the surface of a T cell. In particular, the T cell population can be stimulated as described herein, such as by contact with an anti-CD3 antibody or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or with a protein kinase C activating factor ( For example, bryozolidin) and calcium ionophore contact. To co-stimulate the accessory molecule on the surface of the T cell, a ligand that binds to the helper molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions suitable for stimulating T cell proliferation. To stimulate proliferation of CD4+ T cells or CD8+ T cells, anti-CD3 antibodies and anti-CD28 antibodies can be used. Examples of anti-CD28 antibodies include 9.3, B-T3, XR-CD28 (Diaclone, Besançon, France), which can be used in the same manner as other methods generally known in the art (Berg et al., Transplant Proc. 30(8): 3975 -3977, 1998; Haanen et al, J. Exp. Med. 190(9): 13191328, 1999; Garland et al, J. Immunol Meth. 227 (1-2): 53-63, 1999).

In some aspects, the primary stimulation signals and costimulatory signals for T cells can be provided by different protocols. For example, the agent that provides each signal can be in solution or coupled to a surface. When coupled to a surface, the agent can be coupled to the same surface (ie, "shun" form) or to a different surface (ie, "reverse" form). Alternatively, one agent can be coupled to the surface and the other agent is in solution. In one aspect, an agent that provides a costimulatory signal binds to the cell surface and the agent that provides the primary activation signal is in solution or coupled to the surface. In some aspects, both agents can be in solution. In one aspect, the agents may be in a soluble form The formula is then cross-linked to a surface (such as a cell or antibody that expresses an Fc receptor or other binding agent that will bind to the agent). In this regard, regarding artificial antigen presenting cells (aAPC), for example, U.S. Patent Application Publication Nos. 20040101519 and No. 20060034810, which are intended to be used for activating and expanding T cells in the present invention.

In one aspect, the two agents are immobilized on the beads (the same bead, ie "shun"; or different beads, ie "reverse"). For example, the agent that provides the primary activation signal as an anti-CD3 antibody or antigen-binding fragment thereof and provides a costimulatory signal is an anti-CD28 antibody or antigen-binding fragment thereof; and the two agents are fixed together in the same molecular weight Beads. In one aspect, a 1:1 ratio of each antibody bound to the beads was used for CD4+ T cell expansion and T cell growth. In certain aspects of the invention, a certain ratio of anti-CD3:CD28 antibodies bound to the beads is used such that the expansion of T cells is increased compared to the amplification observed using a 1:1 ratio. In one particular aspect, an increase from about 1 to about 3 times was observed compared to the amplification observed using a 1:1 ratio. In one aspect, the ratio of CD3:CD28 antibodies bound to the beads ranges from 100:1 to 1:100 and all integer values therebetween. In one aspect of the invention, the anti-CD28 antibody bound to the particle is more than the anti-CD3 antibody, i.e., the ratio of CD3:CD28 is less than one. In certain aspects of the invention, the ratio of anti-CD28 antibody bound to the beads to the anti-CD3 antibody is greater than 2:1. In a particular aspect, a 1:100 CD3:CD28 ratio of antibody bound to the beads was used. In one aspect, a 1:75 CD3:CD28 ratio of antibody bound to the beads was used. In another aspect, a 1:50 CD3:CD28 ratio of antibody bound to the beads is used. In one aspect, a 1:30 CD3:CD28 ratio of antibody bound to the beads was used. In one aspect, a 1:10 CD3:CD28 ratio of antibody bound to the beads was used. In one aspect, a 1:3 CD3:CD28 ratio of antibody bound to the beads was used. In another aspect, a 3:1 CD3:CD28 ratio of antibody bound to the beads is used.

T cells or other target cells can be stimulated using a ratio of cells from 1:500 to 500:1 and any integer value therebetween. As the average person can easily understand, the ratio of particles to cells It can be determined relative to the particle size of the target cells. For example, small size beads can bind only a few cells, while larger beads can bind to many cells. In some aspects, the ratio of cells to particles is in the range of 1:100 to 100:1 and any integer value therebetween and in other aspects, including from 1:9 to 9:1 and any integer value therebetween. The ratio can also be used to stimulate T cells. The ratio of anti-CD3 and anti-CD28 coupling particles to T cells that cause T cell stimulation can vary as described above, however some preferred values include 1:100, 1:50, 1:40, 1:30, 1: 20, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1 and 15:1, one of the preferred ratios is at least 1:1 particle ratio T cells. In one aspect, a ratio of particles to cells of 1:1 or smaller is used. In a particular aspect, the preferred particle:cell ratio is 1:5. In other aspects, the ratio of particles to cells may vary depending on the number of days of stimulation. For example, in one aspect, the ratio of particles to cells on the first day is 1:1 to 10:1, and thereafter additional particles are added to the cells every day or every other day until 10 days, with a final ratio of 1:1 to 1:10 (based on the number of cells added on the day). In one particular aspect, the ratio of particles to cells on the first day of stimulation was 1:1 and was adjusted to 1:5 on the third and fifth days of stimulation. In one aspect, the particles are added daily or every other day until the final ratio of the first day is 1:1, and the third and fifth days of stimulation are 1:5. In one aspect, the ratio of particles to cells on the first day of stimulation was 2:1 and the stimulation was adjusted to 1:10 on the third and fifth days. In one aspect, the particles are added daily or every other day until the final ratio of the first day is 1:1, and the third and fifth days of stimulation are 1:10. Those skilled in the art will appreciate that a variety of other ratios are available for use in the present invention. In particular, the ratio will vary depending on the particle size as well as the size and type of the cells. In one aspect, the most typical ratios used on the first day are about 1:1, 2:1, and 3:1.

In other aspects of the invention, cells (such as T cells) are combined with the coated beads, followed by separation of the beads from the cells, followed by culturing of the cells. In an alternative aspect, the coated beads are not separated from the cells prior to incubation, but are cultured together. In another aspect, the beads and cells are first concentrated by the application of a force (such as a magnetic force), resulting in cell surface markers. The junction of the substances is increased, thereby inducing cell stimulation.

For example, cell surface proteins can be joined by contacting paramagnetic beads (3 x 28 beads) with anti-CD3 and anti-CD28 attached to T cells. In one aspect, cells (eg, 10 ⁴ to 10 ⁹ T cells) and beads are combined in a buffer such as PBS (without divalent cations such as calcium and magnesium) (eg, a 1:1 ratio of DYNABEADS®) M-450 CD3/CD28 paramagnetic beads). In addition, it is easy for a person of ordinary skill to understand any cell concentration that can be used. For example, the target cells in the sample may be minimal and only account for 0.01% of the sample, or all samples (ie, 100%) may contain relevant target cells. Therefore, any cell number is in the context of the present invention. In some aspects, it may be desirable to significantly reduce the volume of particles mixed with the cells (i.e., increase cell concentration) to ensure that the cells are in maximum contact with the particles. For example, in one aspect, about 10,000,000,000 cells/ml, 9,000,000,000 cells/ml, 8,000,000,000 cells/ml, 7,000,000,000 cells/ml, 6,000,000,000 cells/ml, 5,000,000,000 cells/ml or 2,000,000,000 are used. Cell/ml concentration. In one aspect, greater than 100,000,000 cells/ml are used. In another aspect, 10,000,000, 15,000,000, 20,000,000, 25,000,000, 30,000,000, 35,000,000, 40,000,000, 45,000,000 or 50,000,000 cells/ml are used. In another aspect, a concentration of 75,000,000, 80,000,000, 85,000,000, 90,000,000, 95,000,000 or 100,000,000 cells/ml is used. In other aspects, a concentration of 125,000,000 or 150,000,000 cells/ml can be used. Use of high concentrations can result in increased cell yield, cell activation, and cell expansion. Furthermore, the use of high cell concentrations allows for more efficient capture of cells that may be weakly associated with a target antigen, such as CD28-negative T cells. Such cell populations can have therapeutic value and will need to be obtained in certain aspects. For example, the use of high concentration cells allows for more efficient selection of CD8+ T cells that typically have weaker CD28 expression.

In one embodiment, a nucleic acid transduced cell encoding a CAR, such as a CAR described herein, is amplified, for example, by the methods described herein. In one embodiment, cell culture expansion Increase hours (eg, about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 18, 21 hours) to about 14 days (eg 1, 2, 3, 4, 5, 6, 7) Time period of 8, 8, 10, 11, 12, 13 or 14 days). In one embodiment, the cells are expanded for a period of from 4 to 9 days. In one embodiment, the cells are expanded for a period of 8 days or less, such as a period of 7, 6, or 5 days. In one embodiment, cells, such as the BCMA CAR cells described herein, are cultured for 5 days and the resulting cells are more potent than the same cells cultured for 9 days under the same culture conditions. Efficacy can be defined, for example, by various T cell functions, such as proliferation, target cell killing, cytokine production, activation, migration, or a combination thereof. In one embodiment, cells that are expanded for 5 days, such as the BCMA CAR cells described herein, exhibit at least one, two, at the time of antigen stimulation, compared to the same cells cultured for 9 days under the same culture conditions. Three or four times. In one embodiment, the cells, such as cells expressing BCMA CAR as described herein, are cultured for 5 days of culture, and the resulting cells exhibit proinflammatory cells as compared to the same cells cultured for 9 days under the same culture conditions. Factor production, such as higher levels of IFN-[gamma] and/or GM-CSF. In one embodiment, cells that are expanded for 5 days, such as the BCMA CAR cells described herein, exhibit pro-inflammatory cytokine production, such as IFN-, compared to the same cells cultured for 9 days under the same culture conditions. The gamma and/or GM-CSF content is increased by at least one, two, three, four, five, ten or more times (in pg/ml).

In one aspect of the invention, the mixture can be cultured for a number of hours (about 3 hours) to about 14 days or any hourly integer value therebetween. In one aspect, the mixture can be cultured for 21 days. In one aspect of the invention, the beads and T cells are cultured together for about eight days. In one aspect, the beads are incubated with T cells for 2-3 days. It may also be desirable to have several stimulation cycles such that the T cell culture time can be 60 days or longer. Suitable conditions for T cell culture include suitable media (eg, minimal medium or RPMI medium 1640 or X-vivo 15 (Lonza)), including serum (eg, fetal bovine serum or human serum), which may be necessary for proliferation and viability. Interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFβ and TNF-α or familiar with this item Any other additive for cell growth is known to the skilled person. Other additives for cell growth include, but are not limited to, surfactants, human plasma protein powders, and reducing agents such as N-ethinyl-cysteine and 2-mercaptoethanol. The medium may include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15 and X-Vivo 20, Optimizer, with the addition of amino acids, sodium pyruvate and vitamins, serum-free or supplemental There is an appropriate amount of serum (or plasma) or a defined group of hormones, and / or a sufficient amount of cytokines for T cell growth and expansion. Antibiotics (eg, penicillin and streptomycin) are included only in the experimental culture and not in the cell culture to be infused into the individual. Target cells were maintained in growth conditions necessary for the support, for example an appropriate temperature (e.g. 37 [deg.] C) and atmosphere (e.g., air plus 5% CO _2).

In one embodiment, the cells are expanded in a suitable medium (eg, a medium described herein) comprising one or more interleukins such that, for example, as measured by methods described herein, such as flow cytometry, The cells were increased by at least 200-fold (eg, 200-fold, 250-fold, 300-fold, 350-fold) during the 14-day expansion period. In one embodiment, the cells are expanded in the presence of IL-15 and/or IL-7 (eg, IL-15 and IL-7).

In embodiments, the methods described herein, eg, methods of producing cells expressing CAR, comprise, for example, removal of T regulatory cells, eg, CD25+, from a population of cells using an anti-CD25 antibody or fragment thereof or a CD25 binding ligand IL-2. T cells. Methods for removing T regulatory cells, such as CD25+ T cells, from a population of cells are described herein. In embodiments, the methods, eg, methods of manufacture, further comprise a population of cells (eg, a population of cells in which T regulatory cells, such as CD25+ T cells have been depleted; or have been pre-contacted with an anti-CD25 antibody, a fragment thereof, or a CD25 The cell population of the binding ligand is contacted with IL-15 and/or IL-7. For example, a population of cells (eg, having been pre-contacted with an anti-CD25 antibody, a fragment thereof, or a CD25 binding ligand) is amplified in the presence of IL-15 and/or IL-7.

In some embodiments, during the production of cells expressing CAR, such as ex vivo, the cells expressing CAR as described herein are comprised of a protein comprising interleukin-15 (IL-15) polypeptide, interleukin-15 receptor alpha ( IL-15Ra) polypeptide or a combination of an IL-15 polypeptide and an IL-15Ra polypeptide (eg, hetIL-15) The composition is in contact. In an embodiment, the CAR-expressing cells described herein are contacted with a composition comprising an IL-15 polypeptide during manufacture of cells expressing CAR, such as ex vivo. In an embodiment, the CAR-expressing cells described herein are contacted with a composition comprising a combination of an IL-15 polypeptide and an IL-15 Ra polypeptide during production of cells expressing CAR, such as ex vivo. In an embodiment, the CAR-expressing cells described herein are contacted with a composition comprising hetIL-15 during the production of cells expressing CAR, such as ex vivo.

In one embodiment, the CAR-expressing cells described herein are contacted with a composition comprising hetIL-15 during ex vivo expansion. In one embodiment, the CAR-expressing cells described herein are contacted with a composition comprising an IL-15 polypeptide during ex vivo expansion. In one embodiment, the CAR-expressing cells described herein are contacted with a composition comprising an IL-15 polypeptide and an IL-15Ra polypeptide during ex vivo amplification. In one embodiment, the contacting causes lymphocyte subpopulations, such as CD8+ T cells, to survive and proliferate.

T cells that have been exposed to different stimulation times can display different characteristics. For example, a typical blood or spherical peripheral blood mononuclear cell product has a population of helper T cells (TH, CD4+) that is greater than the cytotoxic or suppressor T cell population (TC, CD8+). Ex vivo expansion of T cells by stimulation of the CD3 and CD28 receptors produces a population of T cells consisting primarily of TH cells from about day 8 to day 9, whereas after about day 8 to day 9, T cell populations Contains a growing population of TC cells. Thus, depending on the purpose of the treatment, it may be advantageous to infuse the individual with a population of T cells that primarily comprise TH cells. Similarly, if an antigen-specific subset of TC cells has been isolated, it is desirable to amplify this subset to a greater extent.

Furthermore, in addition to the CD4 and CD8 markers, other phenotypic markers vary significantly, but most are reproducible during the cell expansion process. Thus, such reproducibility enables the adjustment of activated T cell products for specific purposes.

Once BCMA CAR is constructed, a variety of assays can be used to assess molecular activity, such as, but not limited to, the ability to expand T cells after antigen stimulation, support T cell expansion in the absence of re-stimulation, and appropriate in vitro and in vivo animals. Anticancer activity in the model. Detailed description below Analytical method for assessing the role of BCMA CAR.

Western blot analysis of CAR expression in primary T cells can be used to detect the presence of monomers and dimers. See, for example, Milone et al, Molecular Therapy 17(8): 1453-1464 (2009). Briefly, T cells expressing CAR (a 1:1 mixture of CD4 ⁺ and CD8 ⁺ T cells) were expanded in vitro for more than 10 days, followed by cell lysis and SDS-PAGE under reducing conditions. The TCR-ζ chain antibody was used to detect CAR containing the full-length TCR-ζ cell plastid and the endogenous TCR-ζ chain by Western blotting. The same T cell subset was used for SDS-PAGE analysis under non-reducing conditions to allow for the assessment of covalent dimer formation.

In vitro expansion of CAR ⁺ T cells after antigen stimulation can be measured by flow cytometry. For example, a mixture of CD4 ⁺ and CD8 ⁺ T cells is stimulated with αCD3/αCD28aAPC and subsequently transduced with a lentiviral vector expressing GFP under the control of the promoter to be analyzed. Exemplary promoters include the CMV IE gene, EF-1 alpha, ubiquitin C or phosphoglycerate kinase (PGK) promoters. GFP fluorescence in the CD4 ⁺ and/or CD8 ⁺ T cell subsets was assessed by flow cytometry on day 6 of culture. See, for example, Milone et al, Molecular Therapy 17(8): 1453-1464 (2009). Alternatively, a magnetic bead coated with αCD3/αCD28 was used to stimulate a mixture of CD4 ⁺ and CD8 ⁺ T cells on day 0, and a bicistronic lentiviral vector expressing CAR was used on day 1 and 2A ribosome was used. The sequence of eGFP was transduced with CAR. After washing, the culture is re-stimulated with cells expressing BCMA, such as multiple myeloma cell lines or K562-BCMA. 100 IU/ml exogenous IL-2 was added to the culture every other day. GFP ⁺ T cells were enumerated by flow cytometry using bead-based counts. See, for example, Milone et al, Molecular Therapy 17(8): 1453-1464 (2009).

Long-lasting CAR ⁺ T cell expansion in the absence of re-stimulation can also be measured. See, for example, Milone et al, Molecular Therapy 17(8): 1453-1464 (2009). Briefly, the amount of Coulter Multisizer III particle counter, Nexcelom Cellometer Vision or Millipore Scepter was used on Day 8 after stimulation with magnetic beads coated with αCD3/αCD28 on Day 0 and after transduction with the indicated CAR on Day 1. The mean T cell volume (fl) was measured.

Animal models can also be used to measure CART activity. For example, a xenograft model of primary human multiple myeloma in immunodeficient mice treated with human BCMA-specific CAR ⁺ T cells can be used. See, for example, Milone et al, Molecular Therapy 17(8): 1453-1464 (2009). In short, after the MM was established, the mice were randomized to the treatment group. Different numbers of BCMA CART cells can be injected into immunodeficient mice loaded with MM. Animal disease progression and tumor burden were assessed at weekly intervals. The survival curves of the groups were compared using a log-rank test. In addition, the absolute peripheral blood CD4 ⁺ and CD8 ⁺ T cell counts at 4 weeks after T cell injection in immunodeficient mice can also be analyzed. Mice were injected with multiple myeloma cells and after 3 weeks were engineered to express T cell injection of BCMA CAR, for example, by a bicistronic lentiviral vector encoding a CAR linked to eGFP. T cells were corrected for 45-50% of the input GFP ⁺ T cells by mixing with mock transduced cells prior to injection and confirmed by flow cytometry. Animal leukemia was assessed at 1-week intervals. The survival curves of the CAR ⁺ T cell group were compared using a time series test.

Evaluation of cell proliferation and cytokine production has been previously described, for example, Milone et al, Molecular Therapy 17(8): 1453-1464 (2009). Briefly, CAR-mediated proliferation or other BCMA-producing myeloma cells were assessed to be irradiated with gamma radiation prior to use by mixing washed T cells with K562 cells expressing BCMA in a microtiter plate. Anti-CD3 (pure line OKT3) and anti-CD28 (pure line 9.3) monoclonal antibodies were added to cultures with KT32-BBL cells for use as a positive control for T cell proliferation, so these signals support long-term CD8 ⁺ T cells. In vitro amplification. As described by the manufacturer using fluorescent beads CountBright ^TM (Invitrogen, Carlsbad, CA) and flow cytometry cytometry measurements include T cells in culture. CAR ⁺ T cells were identified by GFP expression using T cells engineered with eGFP-2A-linked Lentiviral vector expressing CAR. For CAR+ T cells that did not express GFP, CAR+ T cells were detected using biotinylated recombinant BCMA protein and secondary antibiotic protein-PE conjugate. CD4+ and CD8 ⁺ expression on T cells was also detected using specific monoclonal antibodies (BD Biosciences). Cytokine measurements were taken on supernatants collected after 24 hours of restimulation using the human TH1/TH2 cytokine cytology bead array kit (BD Biosciences, San Diego, CA) according to the manufacturer's instructions. Fluorescence was assessed using a FACScalibur flow cytometer and the data was analyzed according to the manufacturer's instructions.

Cytotoxicity can be assessed by standard 51Cr release assays. See, for example, Milone et al, Molecular Therapy 17(8): 1453-1464 (2009). Briefly, target cells (eg, K562 cell line expressing BCMA and primary multiple myeloma cells) were loaded with 51Cr (eg, NaCrO4, New England Nuclear, Boston, MA) at 37 ° C with frequent agitation. After 2 hours, wash twice in complete RPMI and inoculate into a microtiter plate. In each well, in effector cells, different effector cells: target cell (E:T) ratio of effector T cells and target cells were mixed. Additional wells containing only medium (spontaneous release, SR) or 1% triton-X 100 detergent solution (total release, TR) were also prepared. After incubation at 37 ° C for 4 hours, the supernatant was harvested from each well. The released 51Cr was then measured using a gamma particle counter (Packard Instrument Co., Waltham, MA). Each condition was repeated at least three times, and the percentage of dissolution was calculated using the following formula: % dissolution = (ER-SR) / (TR-SR), where ER represents the average 51Cr released for each experimental condition.

Imaging techniques can be used to assess the specific transport and proliferation of CAR in tumor-loaded animal models. Such analysis has been described, for example, in Barrett et al., Human Gene Therapy 22: 1575-1586 (2011). Briefly, NOD/SCID/γc ^-/- (NSG) mice or other immunodeficient mice were injected IV with multiple myeloma cells, followed by 7 days after BCMA with electroporation of CAR constructs for 4 hours. Injection of CART cells. T cells were stably transfected with lentiviral constructs to express firefly luciferase and for bioluminescence, mice were imaged. Alternatively, the therapeutic efficacy and specificity of a single injection of CAR ⁺ T cells in a multiple myeloma xenograft model can be measured as follows: NSG mice are transduced to stabilize multiple myeloma cells expressing firefly luciferase After the injection, T cells were electroporated by the BCMA CAR construct in a single tail vein several days later. Animals were imaged at various time points after injection. For example, a photon density heat map of firefly luciferase positive tumors in representative mice on day 5 (2 days prior to treatment) and day 8 (24 hours after CAR ⁺ PBL) can be generated.

Alternatively, or in combination with the methods disclosed herein, methods and compositions for one or more of: detecting and/or quantifying cells expressing CAR (eg, in vitro or in vivo (eg, clinical monitoring)); immunization Cell expansion and/or activation; and/or CAR specific selection, including the use of CAR ligands. In an exemplary embodiment, the CAR ligand is an antibody that binds to a CAR molecule, eg, binds to an extracellular antigen binding domain of a CAR (eg, an antibody that binds to an antigen binding domain, eg, an anti-idiotypic antibody; or An antibody that binds to the constant region of the extracellular binding domain). In other embodiments, the CAR ligand is a CAR antigen molecule (eg, a CAR antigen molecule as described herein).

In one aspect, a method for detecting and/or quantifying cells expressing CAR is disclosed. For example, CAR ligands can be used to detect and/or quantify cells expressing CAR (eg, clinical monitoring of cells expressing CAR in a patient, or patient administration) in vitro or in vivo. The method comprises: providing a CAR ligand (as appropriate, labeled CAR ligand, eg, a CAR ligand comprising a tag, a bead, a radioactive or fluorescent label); acquiring a cell expressing the CAR (eg obtaining a performance-containing CAR) A sample of the cells, such as a sample or a clinical sample; the CAR-expressing cells are contacted with the CAR ligand under conditions in which binding occurs, thereby detecting the amount (eg, amount) of cells present in the presence of CAR. Binding of cells expressing CAR to CAR ligands can be detected using standard techniques such as FACS, ELISA, and the like.

In another aspect, a method of amplifying and/or activating cells (eg, immune effector cells) is disclosed. The method comprises: providing a cell expressing CAR (eg, a cell that first expresses CAR or a short-lived CAR) The cells expressing the CAR are contacted with a CAR ligand (eg, a CAR ligand as described herein) under conditions in which expansion and/or proliferation of immune cells occurs, thereby producing activation and/or Amplified cell population.

In certain embodiments, a CAR ligand is present (eg, immobilized or attached) to a substrate, such as a non-naturally occurring receptor. In some embodiments, the substrate is a non-cellular substrate. The non-cellular substrate can be a solid support selected from the group consisting of, for example, a disk (eg, a microtiter plate), a membrane (eg, a nitrocellulose membrane), a matrix, a wafer, or a bead. In an embodiment, the CAR ligand is present in the substrate (eg, on the surface of the substrate). The CAR ligand can be immobilized, attached, or covalently or non-covalently associated (eg, cross-linked) to the substrate. In one embodiment, the CAR ligand is attached (eg, covalently attached) to the beads. In the foregoing embodiments, the population of immune cells can be expanded in vitro or ex vivo. The method can further comprise culturing the population of immune cells in the presence of a ligand of the CAR molecule, for example, using any of the methods described herein.

In other embodiments, the method of amplifying and/or activating cells further comprises adding a second stimulatory molecule, such as CD28. For example, the CAR ligand and the second stimulatory molecule can be immobilized to a substrate, such as one or more beads, thereby providing increased cell expansion and/or activation.

In yet another aspect, a method of selecting or enriching cells expressing CAR is provided. The method comprises contacting a CAR-expressing cell with a CAR ligand as described herein; and selecting a cell based on the binding of the CAR ligand.

In other embodiments, a method of depleting, reducing, and/or killing cells expressing CAR is provided. The method comprises contacting a CAR-expressing cell with a CAR ligand as described herein; and targeting the cell based on the binding of the CAR ligand, thereby reducing the number of cells expressing the CAR and/or killing the cell expressing CAR . In one embodiment, the CAR ligand is coupled to a toxic agent (eg, a toxin or a cell ablation drug). In another embodiment, an anti-idiotypic antibody can elicit an effector cell activity, such as ADCC or ADC activity.

Exemplary anti-CAR antibodies that can be used in the methods disclosed herein are described, for example, in WO 2014/190273 and Jena et al., "Chimeric Antigen Receptor (CAR) - Specific Monoclonal Antibody to Detect CD 19 - Specific T cells in Clinical Trials", PLOS March 2013 8:3 e57838 (content incorporated by reference). In one embodiment, the anti-idiotypic antibody molecule recognizes an anti-CD19 antibody molecule, such as an anti-CD19 scFv. For example, an anti-individual genotype antibody molecule can compete with the CD19-specific CAR mAb pure line 136.20.1 as described in Jena et al., PLOS March 8, 8:3 e57838; may have a CD m-specific CAR mAb Pure CDRs of the same No. 136.20.1 (eg, using Kabat definition, Chothia definition or a combination of Kabat and Chothia definitions, one or more of VH CDR1, VH CDR2, CH CDR3, VL CDR1, VL CDR2 and VL CDR3, eg All;; may have one or more (eg, 2) variable regions such as CD19-specific CAR mAb pure line 136.20.1, or may comprise CD19-specific CAR mAb pure line 136.20.1. In some embodiments, the anti-idiotypic antibody is prepared according to the methods described in Jena et al. In another embodiment, the anti-idiotypic antibody molecule is an anti-idiotypic antibody molecule as described in WO 2014/190273. In some embodiments, the anti-idiotypic antibody molecule has the same CDR as the antibody molecule of WO 2014/190273, such as 136.20.1 (eg, in VH CDR1, VH CDR2, CH CDR3, VL CDR1, VL CDR2, and VL CDR3) One or more, for example all); may have one or more (eg 2) variable regions of the antibody molecule of WO 2014/190273, or may comprise an antibody molecule of WO 2014/190273, such as 136.20.1. In other embodiments, the anti-CAR antibody binds to, for example, the constant region of the extracellular binding domain of a CAR molecule as described in WO 2014/190273. In some embodiments, the anti-CAR antibody binds to a constant region of an extracellular binding domain of a CAR molecule, such as a heavy chain constant region (eg, a CH2-CH3 hinge region) or a light chain constant region. For example, in some embodiments, an anti-CAR antibody competes for binding to a 2D3 monoclonal antibody described in WO 2014/190273, having the same CDR as 2D3 (eg, VH CDR1, VH CDR2, CH CDR3, VL CDR1, VL One or more of CDR2 and VL CDR3, for example all), or a variable region having one or more (eg, 2) 2D3, or 2D3 as described in WO 2014/190273.

In some aspects and embodiments, optimization of a particular subset of T cells is described, for example, as described in U.S. Patent No. 62/031,699, the disclosure of which is incorporated herein by reference in its entirety. Compositions and methods herein. In some embodiments, the optimized T cell subset exhibits enhanced persistence compared to control T cells, such as different types (eg, CD8 ⁺ or CD4 ⁺ ) T cells that exhibit the same construct.

In some embodiments, the CD4 ⁺ T cells comprise a CAR as described herein, the CAR comprising an intracellular signaling domain, such as an ICOS domain, suitable for (eg, optimized for enhanced persistence) CD4 ⁺ T cells. In some embodiments, CD8 ⁺ T cells as described herein comprising the CAR, CAR which is adapted to contain (e.g. persistence be optimized for enhanced) CD8 ⁺ T cells in the intracellular signaling domain, e.g. 4-1BB structure Domain, CD28 domain or another costimulatory domain other than the ICOS domain. In some embodiments, a CAR described herein comprises an antigen binding domain as described herein, eg, a CAR comprising an antigen binding domain that targets BCMA, such as the CAR of Table 1 or 16.

In one aspect, described herein is a method of treating an individual, such as an individual having cancer. The method comprises administering to the individual an effective amount: 1) CD4 ⁺ T cells comprising CAR (CAR ^CD4+ ): the CAR comprises: an antigen binding domain, such as an antigen binding domain as described herein, eg, targeting BCMA An antigen binding domain, such as the antigen binding domain of Table 1 or 16; a transmembrane domain; and an intracellular signaling domain, such as a first costimulatory domain, such as an ICOS domain; and 2) a CAR (CAR ^CD8+) a CD8 ⁺ T cell comprising: an antigen binding domain, such as an antigen binding domain as described herein, eg, an antigen binding domain that targets BCMA, such as the antigen binding domain of Table 1 or 16; a transmembrane structure And an intracellular signaling domain, such as a second costimulatory domain, such as a 4-1BB domain, a CD28 domain, or another costimulatory domain other than the ICOS domain; wherein CAR ^CD4+ and CAR ^CD8+ are different from each other.

Optionally, the method further comprises administering: 3) a second CD8+ T cell comprising CAR (second CAR ^CD8+ ), the CAR comprising: an antigen binding domain, such as an antigen binding domain as described herein, eg, specific binding An antigen binding domain of BCMA, such as the antigen binding domain of Table 1 or 16; a transmembrane domain; and an intracellular signaling domain, wherein the second CAR ^CD8+ comprises an intracellular signaling structure not present on CAR ^CD8+ Domains, such as co-stimulatory signaling domains, and optionally do not contain an ICOS signaling domain.

Other assays, including the assays described in the Examples section herein and assays known in the art, can also be used to evaluate the BCMA CAR constructs of the present invention.

Treatment application BCMA related diseases and / or conditions

In one aspect, the invention provides methods for treating a disease associated with BCMA performance. In one aspect, the invention provides a method for treating a condition in which a portion of the tumor is BCMA negative and a portion of the tumor is BCMA positive. For example, the CAR of the present invention can be used to treat an individual who has been treated for a disease associated with elevated BCMA performance, wherein the individual who has been treated for an elevated BCMA level exhibits a disease associated with elevated BCMA levels. In embodiments, the CAR of the invention can be used to treat an individual who has been treated for a disease associated with BCMA performance, wherein the individual who has been treated in association with BCMA performance exhibits a disease associated with BCMA performance.

In one embodiment, the invention provides a method for treating a condition in which BCMA is expressed on both normal cells and cancer cells, but on a normal cell. In one embodiment, the method further comprises selecting, for example, an assay as described herein to allow BCMA CAR to bind and kill cancer cells that exhibit BCMA, but kill less than 30%, 25%, 20%, 15 %, 10%, 5% or less of the affinity of the normal cells of BCMA to bind the CAR of the present invention. For example, kill analysis based on Cr51 CTL can be used, such as flow cytometry. In one embodiment, the BCMA CAR has a binding affinity KD of 10 ^-4 M to 10 ^-8 M, for example 10 ^-5 M to 10 ^-7 M, such as 10 ^-6 M or 10 ^-7 M, for the target antigen. Antigen binding domain. In one embodiment, the binding affinity of the BCMA antigen binding domain is at least five fold, ten fold, 20 fold, 30 fold, 50 fold, 100 fold or 1000 fold less than a reference antibody, such as an antibody described herein.

In one aspect, the invention relates to a vector comprising BCMA CAR operably linked to a promoter for expression in a mammalian immune effector cell, such as a T cell or an NK cell. In one aspect, the invention provides a recombinant immune effector cell (eg, a T cell or an NK cell) for use in the treatment of a BCMA CAR of a tumor exhibiting BCMA, wherein the recombinant immune effector cell (eg, a T cell) exhibits BCMA CAR Or NK cells) are referred to as cells expressing BCMA CAR (eg, BCMA CART or NK cells expressing BCMA CAR). In one aspect, a cell of the invention expressing BCMA CAR (eg, BCMA CART or NK cells expressing BCMA CAR) is capable of contacting tumor cells with at least one BCMA CAR of the invention that is expressed on its surface such that BCMA CAR is expressed Cells such as BCMA CART or NK cells expressing BCMA CAR target tumor cells and inhibit tumor growth.

In one aspect, the invention relates to a method of inhibiting growth of a tumor cell expressing BCMA comprising contacting a tumor cell with a cell of the invention expressing BCMA CAR (eg, BCMA CART or NK cells expressing BCMA CAR), Cells expressing BCMA CAR (eg, BCMA CART or NK cells expressing BCMA CAR) are activated in response to an antigen and target cancer cells, wherein tumor growth is inhibited.

In one aspect, the invention relates to a method of treating cancer in an individual. The method comprises administering to the individual a cell of the invention expressing BCMA CAR (eg, BCMA CART or NK cells expressing BCMA CAR) to treat cancer in the subject. An example of a cancer that can be treated by cells of the present invention that express BCMA CAR (e.g., BCMA CART or NK cells expressing BCMA CAR) is a cancer associated with BCMA performance.

The invention encompasses a type of cell therapy wherein immune effector cells (e.g., T cells or NK cells) are genetically modified to express a chimeric antigen receptor (CAR) and express BCMA CAR cells (e.g., BCMA CART or NK expressing BCMA CAR) Cells) are infused into recipients in need. The infused cells are capable of killing tumor cells in the recipient. Unlike antibody therapy, CAR-modified immune effector cells (such as T cells or NK cells) are able to replicate in vivo, producing long-term persistence that can lead to persistent tumor control. In various aspects, the cells (eg, T cells or NK cells) or their progeny administered to the patient remain in the patient for at least four months, five after administration of the cells (eg, T cells or NK cells) to the patient. Month, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, thirteen months, fourteen months, fifteen months, sixteen Month, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 2 years, 3 years, 4 years or 5 year.

The invention also encompasses a type of cell therapy wherein immune effector cells (e.g., T cells or NK cells) are modified, for example, by in vitro transcribed RNA to transiently express a chimeric antigen receptor (CAR) and immune effector cells (e.g., T cells). Or NK cells) are infused into recipients in need. The infused cells are capable of killing tumor cells in the recipient. Thus, in various aspects, the immune effector cells (eg, T cells or NK cells) administered to the patient are present for less than one month after administration of the immune effector cells (eg, T cells or NK cells) to the patient, eg, three weeks, Two weeks, one week.

Without wishing to be bound by any particular theory, the anti-tumor immune response elicited by CAR-modified immune effector cells (eg, T cells or NK cells) may be an active or passive immune response, or It can be attributed to direct immune response versus indirect immune response. In one aspect, CAR-transduced immune effector cells (eg, T cells or NK cells) exhibit specific pro-inflammatory cytokine secretion and potent cytolytic activity against soluble BCMA inhibition in response to human cancer cells expressing BCMA. The bystander kills the effect and mediates the regression of existing human tumors. For example, tumor cells with fewer antigens in a heterogeneous field of a tumor exhibiting BCMA can be readily destroyed indirectly by BCMA-directed immune effector cells (eg, T cells or NK cells), which are BCMA-directed immune effector cells. Adjacent antigen-positive cancer cells have previously been reacted.

In one aspect, the intact human CAR modified immune effector cells (e.g., T cells or NK cells) of the invention can be of the type used for mammalian ex vivo vaccination and/or in vivo therapy. In one aspect, the mammal is a human.

For ex vivo immunization, at least one of the following is performed in vitro prior to administration of the cells to the mammal: i) amplifying the cells, ii) introducing a nucleic acid encoding the CAR into the cells, or iii) cryopreserving the cells.

Ex vivo procedures are well known in the art and are discussed more fully below. Briefly, cells are isolated from a mammal (e.g., a human) and genetically modified (i.e., transduced or transfected in vitro) using a vector that exhibits the CAR disclosed herein. CAR modified cells are administered to mammalian recipients to provide a therapeutic benefit. The mammalian recipient can be human and the CAR modified cells can be autologous to the recipient. Alternatively, the cells may be allogeneic, homologous or heterologous to the recipient.

The procedure for the ex vivo expansion of hematopoietic stem and progenitor cells is described in U.S. Patent No. 5,199,942, which is incorporated herein by reference, and is incorporated herein by reference. Other suitable methods are known in the art, and thus the invention is not limited to any particular method of ex vivo expansion of cells. Briefly, ex vivo culture and expansion of T cells comprises: (1) collecting CD34+ hematopoietic stem and progenitor cells from mammals by peripheral blood collection or bone marrow explants; and (2) amplifying such cells ex vivo. . Cell growth as described in U.S. Patent No. 5,199,942 In addition to long factors, other factors such as flt3-L, IL-1, IL-3 and c-kit ligands can be used to culture and expand such cells.

In addition to the use of cell-based vaccines in ex vivo immunization, the present invention also provides compositions and methods for immunization in vivo to elicit an immune response against an antigen in a patient.

In general, activated and expanded cells as described herein can be used to treat and prevent diseases produced in individuals with immune dysfunction. In particular, the CAR-modified immune effector cells (e.g., T cells or NK cells) of the invention are used to treat diseases, disorders, and conditions associated with BCMA performance. In certain aspects, the cells of the invention are used to treat a patient at risk of developing a disease, disorder, and condition associated with BCMA performance. Accordingly, the invention provides a method for treating or preventing a disease, disorder, and condition associated with BCMA expression comprising administering to a subject in need thereof a therapeutically effective amount of a CAR-modified immune effector cell of the invention (eg, a T cell) Or NK cells).

In one aspect, the CAR-expressing cells of the present invention (eg, CART cells or NK cells expressing CAR) can be used to treat proliferative diseases, such as cancer or malignant diseases, or precancerous conditions, such as myelodysplasia, myeloid development. Adverse syndrome or leukemia prodrosis. In one aspect, the cancer is a blood cancer. The pathology of blood cancer is a type of cancer that affects the blood, bone marrow, and lymphatic system, such as leukemia and malignant lymphoid tissue. In one aspect, the blood cancer is leukemia or blood disease. An example of a disease or condition associated with BCMA is multiple myeloma (also known as MM) (see Claudio et al, Blood. 2002, 100(6): 2175-86; and Novak et al, Blood. 2004, 103 (2): 689-94). Multiple myeloma, also known as plasma cell myeloma or Kahler's disease, is a cancer characterized by abnormal or malignant plasma B cells accumulating in the bone marrow. Often, cancer cells invade adjacent bones, destroying bone structures and causing bone pain and fracture. Most cases of myeloma also involve the production of paraproteins (also known as M proteins or myeloma proteins), which are an abnormal immunoglobulin that is overproduced by the pure proliferation of malignant plasma cells. According to the diagnostic criteria of the International Myeloma Working Group (IMWG), serum mytone levels above 30 g/L are diagnosed as multiple myeloma (see Kyle et al. (2009), Leukemia. 23: 3-9). . Other symptoms or signs of multiple myeloma include impaired renal function or renal failure, skeletal lesions, anemia, hypercalcemia, and neurological symptoms.

Standards for distinguishing multiple myeloma from other plasma cell proliferative disorders have been established by the International Myeloma Working Group (see Kyle et al. (2009), Leukemia. 23: 3-9). All three of the following criteria must be met:

- pure bone marrow plasma cells 10%

- the presence of serum and / or urinary protein (except in patients with true non-secretory multiple myeloma)

- Evidence of end-organ destruction attributable to a fundamental plasma cell proliferative disorder, specifically:

○ Hypercalcemia: serum calcium 11.5mg/100ml

○ Kidney deficiency: serum creatinine>1.73mmol/l

○ anemia: normal blood color, normal red blood cells, hemoglobin value> 2g/100ml, lower than the lower limit of normal, or hemoglobin value <10g/100ml

○ Skeletal lesions: osteolytic lesions, severe osteopenia or pathological rupture.

Other plasma cell proliferative disorders that can be treated by the compositions and methods described herein include, but are not limited to, asymptomatic myeloma (and flaccid myeloma or inert myeloma), gamma-global blood of unknown significance Disease (MGUS), Waldenstrom macroglobulinemia, plasmacytoma (eg plasma cell cachexia, solitary myeloma, solitary plasmacytoma, extramedullary plasmacytoma and multiple plasmacytoma), whole body Amyloid light chain amyloidosis and POEMS syndrome (also known as Crohn-Fox syndrome, sorghum and PEP syndrome).

Two staging systems for the staging of multiple myeloma: the International Staging System (ISS) (see Greipp et al. (2005), J. Clin. Oncol. 23(15): 3412-3420) and Dürer-Saar Durie-Salmon Staging system (DSS) (see Durie et al. (1975), Cancer 36(3): 842-854). The two staging systems are outlined in the following table:

* The Dürer-Salmen staging system also includes sub-categories that indicate the state of renal function. Add the name "A" or "B" after the number of periods, where "A" indicates relatively normal renal function (serum creatinine value <2.0 mg/dL) and B indicates abnormal renal function (serum creatinine value > 2.0 mg/dL) ).

Standard treatments for multiple myeloma and related diseases include chemotherapy, stem cell transplantation (autologous or allogeneic), radiation therapy, and other drug therapies. Common anti-myeloma drugs include alkylating agents (such as bendamustine, cyclophosphamide, and melphalan), proteasome inhibitors (such as bortezomib), and corticosteroids. (eg dexamethasone and prednisone) and immunomodulators (such as thalidomide and lenalidomide or Revlimid®) or any group thereof Hehe. Bisphosphonate drugs are also often administered in combination with standard anti-MM treatments to prevent bone loss. Patients over 65-70 years of age are unlikely to be candidates for stem cell transplantation. In some cases, dual autologous stem cell transplantation is the preferred choice for patients younger than 60 years of age who responded poorly to the first transplant. The compositions and methods of the invention can be administered in combination with any of the treatments currently designated for multiple myeloma.

Another example of a disease or condition associated with BCMA is Hodgkin's lymphoma and non-Hodgkin's lymphoma (see Chiu et al, Blood. 2007, 109(2): 729-39; He et al, J Immunol. 2004, 172(5): 3268-79).

Hodgkin's lymphoma (HL), also known as Hodgkin's disease, is a lymphoid cancer that originates in white blood cells or lymphocytes. The abnormal cells that make up the lymphoma are called Reed-Sternberg cells. In Hodgkin's lymphoma, cancer spreads from one lymph node group to another. Hodgkin's lymphoma can be subdivided into four pathological subtypes based on the Reed-Sdeberger cell morphology and the cellular composition surrounding the Reed-Sdeberger cells (eg, via lymph node detection): nodules Sclerosing HL, mixed cell subtype, lymphocyte or lymphocyte-rich, lymphocyte depletion. Some Hodgkin's lymphomas may also be nodular lymphocytes predominantly Hodgkin's lymphoma, or may be unspecified. Symptoms and signs of Hodgkin's lymphoma include painless swelling of the lymph nodes in the neck, armpits or groin, fever, night sweats, weight loss, fatigue, itching or abdominal pain.

Non-Hodgkin's lymphoma (NHL) contains different groups of blood cancers including any type of lymphoma other than Hodgkin's lymphoma. Subtypes of non-Hodgkin's lymphoma are primarily classified by cell morphology, chromosomal aberrations, and surface markers. NHL subtypes (or NHL-related cancers) include B-cell lymphomas such as, but not limited to, Burkitt's lymphoma, B-cell chronic lymphocytic leukemia (B-CLL), B-cell pre-lymphocytes Leukemia (B-PLL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL) (eg, intravascular large B-cell lymphoma and primary mediastinal B-cell lymphoma), follicular Lymphoma (eg follicular central lymphoma, follicular small lysed cells), hair thin Cellular leukemia, advanced B-cell lymphoma (Burkitt's-like), lymphoplasmacytic lymphoma (Waldenstrom macroglobulinemia), mantle cell lymphoma, marginal zone B-cell lymphoma (eg extranodal edge) B-cell lymphoma or mucosa-associated lymphoid tissue (MALT) lymphoma, marginal zone B-cell lymphoma and spleen marginal B-cell lymphoma), plasmacytoma/myeloma, prodromal B-lymphoblastic leukemia/lymph Tumor (PB-LBL/L), primary central nervous system (CNS) lymphoma, primary intraocular lymphoma, small lymphocytic lymphoma (SLL); and T cell lymphoma such as (but not limited to) ) pleomorphic large cell lymphoma (ALCL), adult T-cell lymphoma/leukemia (eg, mild, chronic, acute, and lymphoma), vascular central lymphoma, vascular immunoblast T-cell lymphoma, skin T Cellular lymphoma (eg, mycosis fungoides, Sezary syndrome, etc.), extranodal natural killer/T-cell lymphoma (nasal type), enteropathic intestinal T-cell lymphoma, large granular lymphocytic leukemia, Precursor T-lymphoblastic lymphoma/leukemia (T-LBL/L), slow T cells Lymphocytic leukemia / prolymphocytic leukemia (T-CLL / PLL) and peripheral T-cell lymphoma, unspecified. Symptoms and signs of Hodgkin's lymphoma include painless swelling of the lymph nodes in the neck, armpits or groin, fever, night sweats, weight loss, fatigue, itching, abdominal pain, cough or chest pain.

Hodgkin's lymphoma is the same as non-Hodgkin's lymphoma and refers to the degree of spread of cancer cells in the body. In stage I, lymphoma cells are in a lymph node group. In stage II, lymphoma cells are present in at least two lymph node groups, but the two groups are on the same side of the diaphragm, or are present in one part of the tissue or organ and in the lymph nodes of the organ on the same side of the diaphragm. In stage III, the lymphoma cells are in the lymph nodes on either side of the diaphragm, or in one of the tissues or organs adjacent to the lymph node group or in the spleen. In stage IV, lymphoma cells are found in at least one part of at least one organ or tissue, or lymphoma cells are in the organ and in lymph nodes on the other side of the diaphragm. In addition to the Roman numeral staging name, the stage can also be described by the letters A, B, E and S, where A refers to a patient without symptoms, B refers to a patient with symptoms, and E refers to a patient outside the lymphatic system. A patient with lymphoma is found in the tissue, and S is a patient in which lymphoma is found in the spleen.

Hodgkin's lymphoma is usually treated with radiation therapy, chemotherapy or hematopoietic stem cell transplantation. The most common treatment for non-Hodgkin's lymphoma is R-CHOP, which consists of four different chemotherapy (cyclophosphamide, doxorubicin, vincristine, and prenisolone). Rituximab (Rituxan®). Other therapies commonly used in the treatment of NHL include other chemotherapeutic agents, radiation therapy, stem cell transplantation (autologous or allogeneic bone marrow transplantation) or biological therapy, such as immunotherapy. Other examples of biotherapeutics include, but are not limited to, rituximab (Rituxan®), tositumomab (Bexxar®), epratuzumab (LymphoCide®), and alemtuzumab (alemtuzumab, MabCampath®). The compositions and methods of the invention can be administered in combination with any treatment currently designated for use in Hodgkin's lymphoma or non-Hodgkin's lymphoma.

BCMA performance is also associated with Waldenstrom's macroglobulinemia (WM), also known as lymphoplasmacytic lymphoma (LPL) (see Elsawa et al, Blood. 2006, 107(7): 2882-8). Waldenstrom's macroglobulinemia was previously thought to be associated with multiple myeloma, but has recently been classified as a subtype of non-Hodgkin's lymphoma. WM is characterized by uncontrolled B cell lymphocyte proliferation, causing anemia and producing an excess of paraprotein or immunoglobulin M (IgM), which thickens blood and causes hyperviscosity syndrome. Other symptoms or signs of WM include fever, night sweats, fatigue, anemia, weight loss, lymphadenopathy or splenomegaly, blurred vision, dizziness, nosebleeds, bleeding gums, abnormal indocyanine, kidney damage or failure, amyloidosis or peripheral neuropathy .

WM's standard treatment consists of chemotherapy specifically with rituximab (Rituxan®). Other chemotherapeutic drugs can be used in combination, such as Leukeran®, Neosar®, Fludara®, cladribine, Leustatin®, and vincristine. And / or thalidomide. Corticosteroids such as prednisone can also be administered in combination with chemotherapy. Plasmapheresis or plasma exchange is often used throughout the treatment of patients to relieve some of the symptoms by removing paraproteins from the blood. In some cases, Stem cell transplantation is an option for some patients.

Another example of a disease or condition associated with BCMA is brain cancer. Specifically, the performance of BCMA has been associated with astrocytoma or glioblastoma (see Deshayes et al, Oncogene. 2004, 23(17): 3005-12; Pelekanou et al, PLoS One. 2013, 8 (12): e83250). Astrocytoma is a tumor produced by astrocytes, a type of glial cell in the brain. Glioblastoma (also known as glioblastoma multiforme or GBM) is the most malignant form of astrocytoma and is considered to be the most advanced stage of brain cancer (stage IV). There are two variants of glioblastoma: giant cell glioblastoma and glial sarcoma. Other astrocytomas include juvenile hairy cell astrocytoma (JPA), myofibrillar astrocytoma, pleomorphic yellow astrocytoma (PXA), and embryonic dysplastic neuroepithelial neoplasia (DNET) And pleomorphic astrocytoma (AA).

Symptoms or signs associated with glioblastoma or astrocytoma include increased pressure in the brain, headache, seizures, memory loss, behavioral changes, body side movements or sensory loss, language dysfunction, cognitive impairment, Visual impairment, nausea, vomiting, and weakness in the arms or legs.

Surgical removal (or culling) of the tumor is standard treatment to remove as much as possible of the neuroglioma without damaging the normal surrounding brain or minimizing its damage. Radiation therapy and/or chemotherapy are often used postoperatively to inhibit and slow the recurrence of disease from any residual cancer or satellite lesions. Radiation therapy includes whole brain radiation therapy (known external beam radiation), targeted three-dimensional conformal radiation therapy, and targeted radionuclides. Chemotherapeutic agents commonly used to treat glioblastoma include temozolomide, gefitinib or erlotinib, and cisplatin. Angiogenesis inhibitors such as Bevacizumab (Avastin®) are also often used in combination with chemotherapy and/or radiation therapy.

Supportive therapy is also often used to relieve neurological symptoms and improve neurological function, and with this article Any of the cancer therapies described are administered in combination. The main supporting agents include anticonvulsants and corticosteroids. Thus, the compositions and methods of the invention can be used in combination with any standard or supportive treatment for the treatment of glioblastoma or astrocytoma.

Non-cancer related diseases and conditions associated with BCMA performance can also be treated by the compositions and methods disclosed herein. Examples of non-cancer related diseases and conditions associated with BCMA performance include, but are not limited to, viral infections; for example, HIV, fungi, such as Cryptococcus neoformans; colonic irritability; ulcerative colitis and conditions associated with mucosal immunity.

The CAR-modified immune effector cells (e.g., T cells or NK cells) of the invention may be administered alone or as a pharmaceutical composition in combination with a diluent and/or other components, such as IL-2 or other cytokines or cell populations. Cast.

The present invention provides compositions and methods for treating cancer. In one aspect, the cancer is a blood cancer, including but not limited to, the blood cancer is leukemia or lymphoma. In one aspect, a CAR-expressing cell of the invention (eg, a CART cell or a CAR-expressing NK cell) can be used to treat cancer and a malignant disease, such as, but not limited to, for example, acute leukemia, including but not limited to, for example, B Acute lymphoblastic leukemia ("BALL"), acute lymphoblastic leukemia ("TALL"), acute lymphoblastic leukemia (ALL); one or more chronic leukemias, including but not limited to, for example, chronic myelogenous leukemia (CML) ), chronic lymphocytic leukemia (CLL); other hematological or hematological conditions, including but not limited to, for example, B-cell pro-lymphocytic leukemia, parental plasmacytoid dendritic cell tumor, primary Lycoma lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative condition, MALT lymphoma, mantle cell lymphoma, Marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell tumor, Waldens Macroglobulinemia and "precursor leukemia disease", the precursor of leukemia disease by bone marrow to produce blood cells invalid (or dysplasia) various blood pathologies United; and similar disorders. Other diseases associated with BCMA performance include (but Not limited to, for example, atypical and/or non-classical cancers, malignant diseases, precancerous conditions, or proliferative diseases that exhibit BCMA.

In embodiments, the compositions described herein are useful for treating diseases including, but not limited to, plasma cell proliferative disorders, such as asymptomatic myeloma (and flaccid myeloma or inert myeloma), of unknown significance Single gamma globulinemia (MGUS), Waldenstrom macroglobulinemia, plasmacytoma (eg plasma cell cachexia, solitary myeloma, solitary plasmacytoma, extramedullary plasmacytoma and multiple Plasmacytoma), systemic amyloid light chain amyloidosis and POEMS syndrome (also known as Cro-Fox syndrome, sorghum and PEP syndrome).

In embodiments, the compositions described herein are useful for treating diseases including, but not limited to, cancer, such as cancers described herein, such as prostate cancer (eg, castration resistant or therapeutic resistant prostate cancer, or metastasis) Sexual prostate cancer), pancreatic cancer or lung cancer.

The invention also provides methods for inhibiting the proliferation of a population of cells expressing BCMA or reducing the population of cells exhibiting BCMA, the methods comprising reacting a cell population comprising cells expressing BCMA with a cell of the invention that binds to cells expressing BCMA - Cells of BCMA CAR (eg, BCMA CART cells or NK cells expressing BCMA CAR) are contacted. In a particular aspect, the invention provides methods for inhibiting proliferation of a population of cancer cells exhibiting BCMA or reducing a population of cancer cells exhibiting BCMA, the methods comprising modulating a population of cancer cells expressing BCMA with cells expressing BCMA The cells of the invention exhibiting anti-BCMA CAR (e.g., BCMA CART cells or NK cells expressing BCMA CAR) are contacted. In one aspect, the invention provides methods for inhibiting proliferation of a population of cancer cells exhibiting BCMA or reducing a population of cancer cells exhibiting BCMA, the methods comprising constituting a population of cancer cells exhibiting BCMA with a cell that binds to cells expressing BCMA The cells of the invention exhibit anti-BCMA CAR (e.g., BCMA CART cells or NK cells expressing BCMA CAR) are contacted. In certain aspects, cells of the invention that exhibit anti-BCMA CAR (eg, BCMA CART cells or NK cells expressing BCMA CAR) cause multiple myeloma Or the number, number, amount or percentage of cells and/or cancer cells in an individual or animal model of a cancer associated with cells expressing BCMA is reduced by at least 25%, at least 30%, at least 40%, at least relative to the negative control. 50%, at least 65%, at least 75%, at least 85%, at least 95% or at least 99% In one aspect, the individual is a human.

The invention also provides methods for preventing, treating and/or managing diseases associated with cells expressing BCMA, such as blood cancers or atypical cancers that exhibit BCMA, the methods comprising administering to a subject in need thereof An anti-BCMA CAR-expressing cell of the present invention (e.g., BCMA CART cells or NK cells expressing BCMA CAR) exhibiting cells of BCMA. In one aspect, the individual is a human. Non-limiting examples of conditions associated with cells expressing BCMA include viral or fungal infections and conditions associated with mucosal immunity.

The present invention also provides methods for preventing, treating and/or managing diseases associated with cells expressing BCMA, the methods comprising administering to a subject in need thereof an expression of the invention-resistant anti-BCMA CAR that binds to cells expressing BCMA Cells (eg BCMA CART cells or NK cells expressing BCMA CAR). In one aspect, the individual is a human.

The present invention provides a method for preventing cancer recurrence associated with cells expressing BCMA, the method comprising administering to a subject in need thereof an anti-BCMA CAR-expressing cell of the present invention (for example, BCMA CART) that binds to a cell expressing BCMA. Cells or NK cells expressing BCMA CAR). In one aspect, the methods comprise administering to an individual in need thereof an effective amount of an anti-BCMA CAR-expressing cell of the invention (eg, BCMA CART cells or NK cells expressing BCMA CAR) that binds to cells expressing BCMA. In combination with an effective amount of another therapy.

Combination therapy

The CAR-expressing cells described herein can be used in combination with other known agents and therapies. As used herein, "combination" administration means that two (or more) different treatments are delivered to an individual during the course of the individual's affliction, for example, after the individual has been diagnosed with the condition And delivering two or more treatments before the condition is cured or eliminated or treatment is stopped for other reasons. In some embodiments, when the second treatment is initially provided, the provision of the first treatment is still present such that there is an overlap in the administration. This is sometimes referred to as "simultaneous" or "parallel delivery" in this article. In other embodiments, the delivery of one treatment ends before another treatment delivery begins. In some embodiments of any of the cases, the treatment is more effective due to combined administration. For example, the second treatment is more effective than the second treatment in the absence of the first treatment, such as using less second treatment to see the same effect, or the second treatment making the symptoms more severe, Or a similar situation can be seen for the first treatment. In some embodiments, the delivery of the parameter that causes the symptom to be alleviated or related to the condition is greater than the parameter observed with a treatment delivery in the absence of another treatment. The effects of the two treatments may be partially additive, fully additive or greater than additive. Delivery may cause the effect of the delivered first treatment to be detectable upon delivery of the second treatment.

The CAR-expressing cells and at least one additional therapeutic agent described herein can be administered simultaneously or sequentially in the same or separate compositions. For sequential administration, the cells expressing CAR described herein can be administered first, and then administered with additional agents, or the order of administration can be reversed.

CAR therapy and/or other therapeutic agents, procedures, or instrumental therapies are administered during the active period of the disease or during the period of disease remission or less active. CAR therapy can be administered prior to, concurrent with, after, or during the course of the condition.

When administered in combination, CAR therapy and other agents (eg, second or third agents) or all agents may be used more than individually, such as the amount or dose of each agent used in monotherapy, high or low, or the same amount or dose. Cast. In certain embodiments, the amount or dose of CAR therapy, other agent (eg, second or third agent), or all agents is used separately, such as the amount or dose of each agent used in monotherapy (eg, at least 20%, at least 30%, at least 40% or at least 50%). In other embodiments, the CAR therapy, other agents (eg, the second or third agent), or all of the agents cause a desired effect (eg, treating cancer) in an amount or dose that is greater than the individual use required to achieve the same therapeutic effect, eg, in a single Therapy use The amount or dose of each agent is low (eg, at least 20%, at least 30%, at least 40%, or at least 50% lower).

In other aspects, the CAR-expressing cells described herein are used in combination with the following treatments: surgery, chemotherapy, radiation, immunosuppressive agents (such as cyclosporine, azathioprine, methotrexate, mycophenolate) Acid esters and FK506), antibodies or other immunosuppressants (such as CAMPATH, anti-CD3 antibodies or other antibody therapies), cytotoxins, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines and irradiation. Peptide vaccines, such as those described in Izumoto et al, 2008 J Neurosurg 108: 963-971.

In certain instances, the compounds of the invention are combined with other therapeutic agents, such as other anticancer agents, antiallergic agents, anti-nausea agents (or anti-vomiting agents), pain relievers, cytoprotective agents, and combinations thereof.

In one embodiment, the first CAR-expressing cell described herein, such as the cells expressing BCMA CAR described herein, can be used in combination with a second CAR-expressing cell. In one embodiment, the second cell expressing CAR comprises a different anti-BMCA binding domain than the anti-BCMA binding domain in the CAR expressed by the first cell expressing the CAR, such as the anti- BCMA binds to the domain's CAR. In one embodiment, the second CAR-expressing cell expresses a CAR comprising an antigen binding domain that targets an antigen other than BCMA (eg, CD19, CD20, CS-1, kappa light chain, CD139, Lewis Y antigen, or CD38) . In one embodiment, the first CAR-expressing cell described herein, such as the cell expressing BCMA CAR described herein, is used in combination with a cell comprising a second manifestation CAR of CD19 CAR. In one embodiment, the cells expressing BCMA CAR described herein are used in combination with cells expressing CD19 CAR to treat a BCMA-associated cancer described herein, such as multiple myeloma. In some embodiments, the multiple myeloma is CD19 negative, such as, for example, flow cytometry, RT-PCR, or flow cytometry and RT-PCR detection, for example, having a majority (eg, at least 98%) 99%, 99.5%, 99.9% or 99.95%) of neoplastic plasma cells with a CD19 negative phenotype. As shown in Example 17 herein, CD19 CAR can also be quite effective against CD19-negative multiple myeloma. While not wishing to be bound by theory, CD19 CAR may act on neonates by targeting cells with a CD19 expression that is less than the detection threshold of the assays described herein or by targeting non-neoplastic cells that support neoplastic cells. A small but important CD19 positive population of cells. In an embodiment, CD19 CAR can remove B cells, such as B regulatory B cells.

For example, in one embodiment, the first CAR-expressing cell described herein, such as a cell that exhibits BCMA CAR, and a second cell of CAR described herein, such as a cell that exhibits CD19 CAR, are prepared in the same manner. The composition is administered at the same time. In another embodiment, the first CAR-expressing cell described herein, such as a cell that exhibits BCMA CAR, and a second cell of CAR that is described herein, such as a cell that exhibits CD19 CAR, is prepared in a separate composition. And separate compositions are administered simultaneously or sequentially. When the cells expressing BCMA CAR and the cells expressing the second CAR are prepared in separate compositions, cells expressing BCMA CAR may be administered first, and secondly, cells expressing CAR may be administered, or the order of administration may be reversed.

In one embodiment, the CD19 CAR is a CD19 CAR as described in WO 2014/153270, which is incorporated herein by reference in its entirety in its entirety, for example, in the the the For example, a sequence of 95%-99% consistent. In some embodiments, the CD19 CAR construct is or is at least 95%, such as 95%-99% identical to the CAR19 construct provided in PCT Publication WO2012/079000, which is incorporated herein by reference in its entirety. sequence. In one embodiment, the anti-CD19 binding domain is the scFv described in WO2012/079000 or a sequence that is at least 95%, such as 95%-99% identical thereto.

In an embodiment, the first cell expressing CAR is administered to the individual and the second cell expressing CAR is administered to the individual. In an embodiment, the first CAR-expressing cell comprises a CAR comprising a CD27 costimulatory domain and a CD3ζ (mutant or wild type) major signaling domain (eg, Such as BCMA or CD19 CAR). In an embodiment, the second CAR-expressing cell comprises a CAR (eg, BCMA CAR) comprising a 4-1BB co-stimulatory domain and a CD3ζ (mutant or wild type) major signaling domain. Without wishing to be bound by theory, in an embodiment, the first cytotoxicity indicative of CAR may be less than that of the second CAR, and is used to reduce tumor volume.

In one embodiment, the CAR-expressing cells described herein can be used in combination with a chemotherapeutic agent. Exemplary chemotherapeutic agents include anthracycline (eg, cranberries (eg, liposomal cranberries)), vinca alkaloids (eg, vinblastine, vincristine, vindesine, vinorelbine) Vinorelbine, alkylating agents (eg cyclophosphamide, decarbazine, melphalan, ifosfamide, temozolomide), immune cell antibodies (eg alemtuzamab) , gemuzumab (gemtuzumab, rituximab, tocilizumab), antimetabolites (including, for example, folic acid antagonists, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors (eg, fluoride) Dalabin)), mTOR inhibitor, TNFR glucocorticoid-induced TNFR-related protein (GITR) agonist, proteasome inhibitor (eg, aclarin A, toxin or bortezomib), immunomodulator ( Such as thalidomide or a thalidomide derivative (such as lenalidomide)).

Chemotherapeutic agents generally considered for combination therapy include anastrozole (Arimidex®), bicalutamide (Casodex®), bleomycin sulfate (Blenoxane®), and white elimination. Busulfan (Myleran®), Busulfex®, capecitabine (Xeloda®), N4-pentyloxycarbonyl-5-deoxy-5-fluorocytosine , carboplatin (Paraplatin®), carmustine (BiCNU®), chlorambucil (Leukeran®), cisplatin (Platinol®), cladribine (Leustatin®), cyclophosphine Indoleamine (Cytoxan® or Neosar®), cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposome injection (DepoCyt®), dacarbazine (dacarbazine) ) (DTIC-Dome®), actinomycin d (dactinomycin) (actinomycin D, Cosmegan), hydrochloride Daunorubicin hydrochloride (Cerubidine®), citric acid citrate liposome injection (DaunoXome®), dexamethasone, docetaxel (Taxotere®), doxorubicin hydrochloride (Adriamycin®, Rubex®), etoposide (Vepesid®), fludarabine phosphate (Fludara®), 5-fluorouracil (Adrucil®, Efudex®), flutamide (Eulexin®), tezacitibine, gemcitabine (difluorodeoxycytidine), hydroxyurea (Hydrea®), Idarubicin (Idamycin®), ifosfamide ( Ifosfamide) (IFEX®), irinotecan (Camptosar®), L-aspartate glutaminase (ELSPAR®), formazan tetrahydrofolate, melphalan (Alkeran®), 6-mercaptopurine ( Purinethol®, Folex®, Novantrone®, mylotarg, paclitaxel (Taxol®), Phoenix (钇90/MX-DTPA) ), pentostatin, Gliadel® with carmustine implants, tamoxifen citrate ( Nolvadex®), teniposide (Vumon®), 6-thioguanine, thiotepa, tirapazamine (Tirazone®), topotecan hydrochloride for injection (Tirzone®) Hycamptin®), Vinban®, Oncovin® and Navelbine®.

Anticancer agents of particular interest in combination with the compounds of the invention include: anthracycline; alkylating agents; antimetabolites; inhibition of calcium-dependent phosphatase calcineurin or p70S6 kinase FK506 or inhibition of p70S6 kinase Drug; mTOR inhibitor; immunomodulator; anthracycline; vinca alkaloid; proteasome inhibitor; GITR agonist; protein tyrosine phosphatase inhibitor; CDK4 kinase inhibitor; BTK inhibitor; MKN kinase Inhibitor; DGK kinase inhibitor; or oncolytic virus.

Exemplary alkylating agents include, but are not limited to, nitrogen mustard, ethyleneimine derivatives, alkyl sulfonates, nitrosoureas, and triazenes: Aminouracil Mustard®, Chlorethaminacil®, Demethyldopan ®, Desmethyldopan®, Haemanthamine®, Nordopan®, Uracil nitrogen mustard®, Uracillost®, Uracilmostaza®, Uramustin®, Uramustine®), Mustargen®, Cytoxan®, Neosar® , Clafen®, Endoxan®, Procytox®, RevimmuneTM ⁾ , Mitoxana®, Alkeran®, Leukeran®, Piper Bromide (Amedel®, Vercyte®), Tritamine (Hemel®, Hexalen®, Hexastat®), Tri-extended ethyl thiophosphoramide, Temodar®, Thioplex®, Busanvex®, Myleran® ), Camexine (BiCNU®), lomustine (CeeNU®), streptozocin (Zanosar®) and dacarbazine (DTIC-Dome®). Additional exemplary alkylating agents include, but are not limited to, Oxaliplatin (Eloxatin®); Temozolomide (Temodar® and Temodal®); Actinomycin d (also known as Actinomycin D, Cosmegen®) ); melphalan (also known as L-PAM, L-lysin and amphetamine, Alkeran®); hexamethylene melamine (also known as hexamethyl melamine (HMM), Hexalen®); BiCNU®; Bendamustine (Treanda®); Busulfim® and Myleran®; Paraplatin®; Lomustine (also known as CCNU, CeeNU®); Cisplatin (also known as CDDP, Platinol® and Platinol®-AQ); Leukeran®; Cytoxan® and Neosar®; Dacarbazine (also known as DTIC, DIC and imidazole carboxamide, DTIC-Dome®); Altretamine (also known as hexamethyl melamine (HMM), Hexalen®); Isoproline (Ifex®); Pride Prednumustine; Procarbazine (Matulane®); Mechlorethamine (also known as nitrogen mustard, mustine and meglumine hydrochloride, Mustargen® ); streptozotocin (Stre) Ptozocin) (Zanosar®); thiotepa (also known as thiophosphonamide, TESPA and TSPA, Thioplex®); cyclophosphamide (Endoxan®, Cytoxan®, Neosar®, Procytox®, Revimumune®); Bendamustine HCl (Treanda®).

Exemplary mTOR inhibitors include, for example, temsirolimus; ridaforolimus (formerly known as deferolimus, dimethylphosphinic acid (1 R , 2 R , 4 S )-4 -[(2 R )-2[(1 R , 9 S , 12 S , 15 R , 16 E , 18 R , 19 R , 21 R , 23 S , 24 E , 26 E , 28 Z , 30 S , 32 S ,35 R )-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-five side Oxyl-11,36-dioxa-4-azatricyclo[30.3.1.0 ^4,9 ]hexa(tricadecane)-16,24,26,28-tetraen-12-yl]propyl] 2-methoxycyclohexyl ester, also known as AP23573 and MK8669, and described in PCT Publication No. WO 03/064383); everolimus (Afinitor® or RAD001); rapamycin (AY22989, Sirolimus®); simapimod (CAS 164301-51-3); emsirolimus, (5-{2,4-bis[(3S)-3-methylmorpholine-4) -yl]pyrido[2,3- d ]pyrimidin-7-yl}-2-methoxyphenyl))methanol (AZD8055); 2-amino-8-[trans-4-(2-hydroxyethyl) Oxy)cyclohexyl]-6-(6-methoxy-3-pyridyl)-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one (PF04691502, CAS 1013101- 36-4); and N ² -[1,4-di-oxy-4-[[4-(4-o-oxy-8-phenyl-4) H -1--1-benzopipene-2-yl)morpholin-4-yl]methoxy]butyl]-L-spermine guanylglycine decyl-L-α-aspartate sulfhydryl L - Serine-(SEQ ID NO: 383), internal salt (SF1126, CAS 936487-67-1) and XL765.

Exemplary immunomodulators include, for example, afutuzumab (available from Roche®); pegfilgrastim (Neulasta®); lenalidomide (CC-5013, Revlimid®); Thalamid®; actimid (CC4047); and IRX-2 (a mixture of human cytokines including interleukin 1, interleukin 2 and interferon gamma, CAS 951209-71-5 , obtained from IRX therapy).

Exemplary anthracyclines include, for example, cranberries (Adriamycin® and Rubex®); bleoxine (lenoxane®); daunorubicin (danomycin hydrochloride, daunorubicin, and erythromycin hydrochloride, Cerubidine®); daunorubicin liposomal (daunorubicin citrate liposome, DaunoXome®); mitoxantrone (DHAD, Novantrone®); epirubicin (Ellence ^TM); idarubicin (Idamycin® , Idamycin PFS®); Mutamycin®; geldanamycin; herbimycin; ravidomycin; and deacetylated lavamycin Desacetylravidomycin).

Exemplary vinca alkaloids include, for example, vinorelbine® (Navelbine®), vincristine (Oncovin®), and vindesine (Eldisine®); vinblastine (also known as vinblastine sulfate, vincaleukoblastine) VLB, Alkaban-AQ® and Velban®); and vinorelbine (Navelbine®).

Exemplary proteasome inhibitors include bortezomib (Velcade®); carbozolium (PX-171-007, ( S )-4-methyl- N -(( S )-1-(((S)-) 4-methyl-1-(( R )-2-methyloxiran-2-yl)-1-oxooxypentan-2-yl)amino)-1-oxo-3-benzene Propionyl-2-yl)-2-(( S )-2-(2-morpholinylacetamido)-4-phenylbutylideneamino)-pentamidine; marizomib (NPI-0052); ixazomib citrate (MLN-9708); delanzomib (CEP-18770); and O -methyl- N -[(2-methyl-) 5-thiazolyl)carbonyl]-L-seramine indolyl- O -methyl- N -[(1 S )-2-[(2 R )-2-methyl-2-oxiranyl]- 2-Sideoxy-1-(phenylmethyl)ethyl]-L-silylamine (ONX-0912).

In the Examples, the CAR-expressing cells described herein are administered to an individual in combination with fludarabine, cyclophosphamide, and/or rituximab. In the Examples, the CAR-expressing cells described herein are administered to a subject in combination with fludarabine, cyclophosphamide, and rituximab (FCR). In an embodiment, the individual has CLL. For example, an individual has a deletion in the short arm of chromosome 17 (del(17p), eg in leukemia cells). In other examples, the individual does not have del(17p). In an embodiment, the individual comprises a leukemia cell comprising a mutation in an immunoglobulin heavy chain variable region ( IgV _H ) gene. In other embodiments, the individual does not comprise leukemia cells comprising a mutation in the immunoglobulin heavy chain variable region ( IgV _H ) gene. In an embodiment, fludarabine is at about 10-50 mg/m ² (eg, about 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, or 45- A dose of 50 mg/m ² ), for example, is administered intravenously. In an embodiment, the cyclophosphamide is administered at a dose of about 200-300 mg/m ² (eg, about 200-225, 225-250, 250-275, or 275-300 mg/m ² ), for example, intravenously. In an embodiment, rituximab of about 400-600mg / m ² (e.g. 400-450,450-500,500-550 or 550-600mg / m ²⁾ dose of, for example, administered intravenously.

In the Examples, the CAR-expressing cells described herein are administered to a subject in combination with bendamustine and rituximab. In an embodiment, the individual has CLL. For example, an individual has a deletion in the short arm of chromosome 17 (del(17p), eg in leukemia cells). In other examples, the individual does not have del(17p). In an embodiment, the individual comprises a leukemia cell comprising a mutation in an immunoglobulin heavy chain variable region ( IgV _H ) gene. In other embodiments, the individual does not comprise leukemia cells comprising a mutation in the immunoglobulin heavy chain variable region (IgV _H ) gene. In an embodiment, the bendamustine is administered at a dose of about 70-110 mg/m ² (eg, 70-80, 80-90, 90-100, or 100-110 mg/m ² ), eg, intravenously. In an embodiment, rituximab of about 400-600mg / m ² (e.g. 400-450,450-500,500-550 or 550-600mg / m ²⁾ dose of, for example, administered intravenously.

In an embodiment, the CAR-expressing cells described herein are administered to an individual in combination with rituximab, cyclophosphamide, cranberry, vincristine, and/or a corticosteroid (eg, prednisone). In the Examples, the CAR-expressing cells described herein are administered to a subject in combination with rituximab, cyclophosphamide, cranberry, vincristine, and prednisone (R-CHOP). In an embodiment, the individual has diffuse large B-cell lymphoma (DLBCL). In an embodiment, the individual has a limited volume of DLBCL (eg, comprising a tumor having a size/diameter of less than 7 cm). In an embodiment, the individual is treated with radiation in combination with R-CHOP. For example, an individual is administered R-CHOP (eg, 1-6 cycles, eg, 1, 2, 3, 4, 5, or 6 R-CHOP cycles) followed by radiation. In some cases, R-CHOP is administered to an individual after radiation (eg, 1-6 cycles, such as 1, 2, 3, 4, 5, or 6 R-CHOP cycles).

In the Examples, the CAR-expressing cells described herein are administered to an individual in combination with etoposide, prednisone, vincristine, cyclophosphamide, cranberry, and/or rituximab. In reality In the examples, the CAR-expressing cells described herein are administered to a subject in combination with etoposide, prednisone, vincristine, cyclophosphamide, cranberry, and rituximab (EPOCH-R). In the Examples, the CAR-expressing cells described herein are administered to a subject in combination with a dose-adjusted EPOCH-R (DA-EPOCH-R). In an embodiment, the individual has a B cell lymphoma, such as Myc rearranged invasive B cell lymphoma.

In an embodiment, the CAR-expressing cells described herein are administered to an individual in combination with rituximab and/or lenalidomide. Lenalidomide ((RS) -3- (4- amino-1-oxo-1,3-dihydro -2 H - isoindol-2-yl) piperidine-2,6-dione) It is an immunomodulator. In the Examples, the CAR-expressing cells described herein are administered to an individual in combination with rituximab and lenalidomide. In an embodiment, the individual has follicular lymphoma (FL) or mantle cell lymphoma (MCL). In an embodiment, the individual has FL and has not previously been treated with cancer therapy. In an embodiment, lenalidomide is administered at a dose of about 10-20 mg (e.g., 10-15 or 15-20 mg), such as daily. In an embodiment, rituximab is at a dose of about 350-550 mg/m ² (eg, 350-375, 375-400, 400-425, 425-450, 450-475, or 475-500 mg/m ² ), For example, intravenous administration.

In the examples, the CAR-expressing cells described herein are administered to an individual in combination with berentuximab. Belenzumab is an antibody-drug conjugate of an anti-CD30 antibody with monomethyl auristatin E. In an embodiment, the individual has Hodgkin's lymphoma (HL), such as relapsed or refractory HL. In an embodiment, the individual comprises CD30+ HL. In an embodiment, the individual has undergone autologous stem cell transplantation (ASCT). In an embodiment, the individual has not performed an ASCT. In an embodiment, the Belenzumab is at a dose of about 1-3 mg/kg (eg, about 1-1.5, 1.5-2, 2-2.5, or 2.5-3 mg/kg), such as intravenously, for example every 3 weeks. Cast.

In the examples, the CAR-expressing cells described herein are administered in combination with bereximab and dacarbazine or in combination with berenztumab and bendamustine. Dacarbazine is an alkylating agent and its chemical name is 5-(3,3-dimethyl-1-triazalyl)imidazole-4-carboxamide. Bendamustine is an alkylating agent with the chemical name 4-[5-[bis(2-chloroethyl)amino]-1-methylbenzimidazol-2-yl]butyric acid. In an embodiment, the individual has Hodgkin's lymphoma (HL). In an embodiment, the individual has not previously been treated with cancer therapy. In an embodiment, the individual is at least 60 years old, such as 60, 65, 70, 75, 80, 85 or older. In an embodiment, dacarbazine is at a dose of about 300-450 mg/m ² (eg, about 300-325, 325-350, 350-375, 375-400, 400-425, or 425-450 mg/m ² ), for example Intravenous administration. In an embodiment, bendamustine about 75-125mg / m ² (e.g. 75-100 or 100-125mg / m ^2, for example from about 90mg / m ²⁾ dose of, for example, administered intravenously. In an embodiment, the Belenzumab is at a dose of about 1-3 mg/kg (eg, about 1-1.5, 1.5-2, 2-2.5, or 2.5-3 mg/kg), such as intravenously, for example every 3 weeks. Cast.

In some embodiments, a CAR-expressing cell described herein is administered to a subject in combination with a CD20 inhibitor, such as an anti-CD20 antibody (eg, an anti-CD20 monospecific antibody or a bispecific antibody) or a fragment thereof. Exemplary anti-CD20 antibodies include, but are not limited to, rituximab, ovalimumab, okuxumab, vitolizumab, ibbituzumab, TRU-015 (Trubion Pharmaceuticals), Ocalazumab (ocaratuzumab) and Pro131921 (Genentech). See, for example, Lim et al. Haematologica. 95.1 (2010): 135-43.

In some embodiments, the anti-CD20 antibody comprises rituximab. Rituximab is a chimeric mouse/human monoclonal antibody 1gG1 kappa that binds to CD20 and causes cytolysis of cells expressing CD20, for example, in www.accessdata.fda.gov/drugsatfda_docs/label/2010/103705s5311lbl.pdf Said. In the Examples, the CAR-expressing cells described herein are administered to a subject in combination with rituximab. In an embodiment, the individual has CLL or SLL.

In some embodiments, rituximab is administered intravenously, for example, as an intravenous infusion. For example, each infusion provides about 500-2000 mg (eg, about 500-550, 550-600, 600-650, 650-700, 700-750, 750-800, 800-850, 850-900, 900-950, 950-1000, 1000-1100, 1100-1200, 1200-1300, 1300-1400, 1400-1500, 1500-1600, 1600-1700, 1700-1800, 1800-1900 or 1900-2000 mg) rituximab. In some embodiments, rituximab is 150 mg/m ² to 750 mg/m ² , such as about 150-175 mg/m ² , 175-200 mg/m ² , 200-225 mg/m ² , 225-250 mg/m. ^{2, 250-300mg / m 2, 300-325mg} / m 2, 325-350mg / m 2, 350-375mg / m 2, 375-400mg / m 2, 400-425mg / m 2, 425-450mg / m 2 , 450-475 mg/m ² , 475-500 mg/m ² , 500-525 mg/m ² , 525-550 mg/m ² , 550-575 mg/m ² , 575-600 mg/m ² , 600-625 mg/m ² , A dose of 625-650 mg/m ² , 650-675 mg/m ² or 675-700 mg/m ² is administered, wherein m ² indicates the body surface area of the individual. In some embodiments, rituximab is administered at a time interval of administration of at least 4 days, such as 4, 7, 14, 21, 28, 35 or more days. For example, rituximab is administered at a dosing interval of at least 0.5 weeks, such as 0.5, 1, 2, 3, 4, 5, 6, 7, 8 or more weeks. In some embodiments, rituximab is administered at a dose and administration interval described herein for a period of time, such as at least 2 weeks, such as at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 weeks or more. For example, rituximab is administered at a dosage and administration interval as described herein, for a total of at least 4 doses per treatment cycle (eg, at least 4, 5, 6, 7, 8, 9, per treatment cycle, 10, 11, 12, 13, 14, 15, 16 or more).

In some embodiments, the anti-CD20 antibody comprises oxalimumab. Autumuzumab is an anti-CD20 IgG1 kappa human monoclonal antibody with a molecular weight of about 149 kDa. For example, ovalimumab is produced using a transgenic mouse and fusion tumor technology and is expressed and purified from a recombinant murine cell line (NS0). See, for example, www.accessdata.fda.gov/drugsatfda_docs/label/2009/125326lbl.pdf; clinical trial identification numbers NCT01363128, NCT01515176, NCT01626352, and NCT01397591. In the examples, the CAR-expressing cells described herein are administered to an individual in combination with ovalimumab. In an embodiment, the individual has CLL or SLL.

In some embodiments, ovalimumab is administered as an intravenous infusion. For example, each infusion provides about 150-3000 mg (eg, about 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, 750-800, 800-850, 850-900, 900- 950, 950-1000, 1000-1200, 1200-1400, 1400-1600, 1600-1800, 1800-2000, 2000-2200, 2200-2400, 2400-2600, 2600-2800 or 2800-3000mg) oxalimumab . In an embodiment, oxacilizumab is administered at an initial dose of about 300 mg, followed by 2000 mg, for example about 11 doses, such as 24 weeks. In some embodiments, ovalimumab is administered at a time interval of administration of at least 4 days, such as 4, 7, 14, 21, 28, 35 or more days. For example, oxalimumab is at least 1 week, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 26, 28, 20, 22, 24, The administration time interval of 26, 28, 30 weeks or more is administered. In some embodiments, oxacilizumab is administered at a dose and administration interval as described herein for a period of time, such as at least 1 week, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 40, 50, 60 weeks or more, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more, or 1, 2, 3, 4, 5 or more years. For example, oxacilizumab is administered at a dose and administration interval as described herein, for a total of at least 2 doses per treatment cycle (eg, at least 2, 3, 4, 5, 6, 7, 8 per treatment cycle) , 9, 10, 11, 12, 13, 14, 15, 16, 18, 20 or more).

In some cases, the anti-CD20 antibody comprises okuxumab. Okuxumab is a humanized anti-CD20 monoclonal antibody, as described, for example, in Clinical Trial Identification Nos. NCT00077870, NCT01412333, NCT00779220, NCT00673920, NCT01194570, and Kappos et al. Lancet. 19.378 (2011): 1779-87.

In some cases, the anti-CD20 antibody comprises vitorizumab. Vitorizumab is a humanized monoclonal antibody against CD20. See, for example, Clinical Trial Identification No. NCT00547066, NCT00546793, NCT01101581, and Goldenberg et al. Leuk Lymphoma. 51(5) (2010): 747-55.

In some cases, the anti-CD20 antibody comprises GA101. GA101 (also known as orbitolizumab or RO5072759) is a humanized and glycosyl engineered anti-CD20 monoclonal antibody. See, for example, Robak. Curr. Opin. Investig. Drugs. 10.6 (2009): 588-96; clinical trial identification numbers: NCT01995669, NCT01889797, NCT02229422, and NCT01414205; and www.accessdata.fda.gov/drugsatfda_docs/label/2013/125486s000lbl .pdf.

In some cases, the anti-CD20 antibody comprises AME-133v. AME-133v (also known as LY2469298 or Ocalaizumab) is a humanized IgG1 monoclonal antibody against CD20, which has increased affinity for FcγRIIIa receptor and antibody-dependent cytotoxicity compared to rituximab. (ADCC) activity is enhanced. See, for example, Robak et al. BioDrugs 25.1 (2011): 13-25; and Forero-Torres et al. Clin Cancer Res. 18.5 (2012): 1395-403.

In some cases, the anti-CD20 antibody comprises PRO131921. PRO131921 is a humanized anti-CD20 monoclonal antibody engineered to better bind to FcyRIIIa and enhance ADCC compared to rituximab. See, for example, Robak et al. BioDrugs 25.1 (2011): 13-25; and Casulo et al. Clin Immunol. 154.1 (2014): 37-46; and clinical trial identification number NCT00452127.

In some cases, the anti-CD20 antibody comprises TRU-015. TRU-015 is an anti-CD20 fusion protein derived from the domain of an antibody against CD20. Although TRU-015 is smaller than the monoclonal antibody, it retains the Fc-mediated effector function. See, for example, Robak et al. BioDrugs 25.1 (2011): 13-25. TRU-015 contains an anti-CD20 single-chain variable fragment (scFv) linked to the human IgG1 hinge, CH2 and CH3 domains but lacking the CH1 and CL domains.

In some embodiments, an anti-CD20 antibody described herein binds or otherwise binds to a therapeutic agent, such as a chemotherapeutic agent (eg, celecoxib, fludarabine, histone deacetylase inhibitor, Methylating agents, peptide vaccines, antitumor antibiotics, tyrosine kinase inhibitors, alkylating agents, anti-microtubule or anti-mitotic agents, anti-allergic agents, anti-nausea agents (or An anti-emetic agent), a pain reliever or a cytoprotective agent as described herein.

In the Examples, the CAR-expressing cells described herein and B-cell lymphoma 2 (BCL-2) inhibitors (eg, Venetoclax, also known as ABT-199 or GDC-0199) and/or rituximab The combination of tricepsin is administered to individuals. In the Examples, the CAR-expressing cells described herein are administered to an individual in combination with Vittoria and Rituximab. Vitolas is a small molecule that inhibits the anti-apoptotic protein BCL-2. The structure of Vitolas is shown below (4-(4-{[2-(4-chlorophenyl))-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazine-1 -yl) -N -({3-nitro-4-[(tetrahydro-2 H -piperidin-4-ylmethyl)amino]phenyl}sulfonyl)-2-(1 H -pyrrole And [2,3- b ]pyridin-5-yloxy)benzamide).

In an embodiment, the individual has CLL. In an embodiment, the individual has recurrent CLL, eg, the individual has previously been administered cancer therapy. In an embodiment, the Vitolas are about 15-600 mg (eg, 15-20, 20-50, 50-75, 75-100, 100-200, 200-300, 300-400, 400-500, or 500-600 mg). The dose, for example, is administered daily. In an embodiment, rituximab is at a dose of about 350-550 mg/m ² (eg, 350-375, 375-400, 400-425, 425-450, 450-475, or 475-500 mg/m ² ), For example, intravenously, for example, monthly administration.

Without being bound by theory, it is believed that in some cancers, B cells (such as B regulatory cells) can inhibit T cells. In addition, the combination of oxaliplatin and B cell depleting agents can reduce tumor size and/or eliminate tumors in an individual. In some embodiments, a CAR-expressing cell (eg, BCMA CAR) described herein is associated with a B cell depleting agent (eg, a cell that exhibits CD19 CAR, a cell that exhibits CD20 CAR, rituximab, okuxumab) , epazumab or belimizumab) and oxaliplatin in combination. In an embodiment, the cancer cell can be CD19 Negative or CD19 positive; or BCMA negative or BMCA positive. In embodiments, a CAR-expressing cell (eg, BCMA CAR) described herein is administered in combination with a B cell depleting agent and oxaliplatin to treat a cancer, such as a cancer described herein, eg, a solid cancer, such as a prostate cancer. , pancreatic cancer or lung cancer.

In embodiments, a CAR-expressing cell (eg, BCMA CAR) described herein can deplete B cells in an individual (eg, having a plasma cell-like phenotype, such as a B cell that exhibits BCMA, CD19, and/or CD20). In an embodiment, the B cell can be CD19 negative or CD19 positive; or BCMA negative or BMCA positive. In some embodiments, the CAR-expressing cells described herein (eg, BCMA CAR) are administered in combination with oxaliplatin. In embodiments, the CAR-expressing cells described herein are administered in combination with oxaliplatin for the treatment of cancer, such as a solid cancer, such as prostate cancer, pancreatic cancer, or lung cancer. In some embodiments, the CAR-expressing cells described herein are administered in combination with an oncolytic virus. In an embodiment, the oncolytic virus is capable of selectively replicating in cancer cells and triggers the death of cancer cells or slows the growth of cancer cells. In some cases, oncolytic viruses have no or minimal effect on non-cancerous cells. Oncolytic viruses include, but are not limited to, oncolytic adenovirus, oncolytic herpes simplex virus, oncolytic retrovirus, oncolytic small virus, oncolytic vaccinia virus, oncolytic Sinbis virus, oncolytic Influenza virus or oncolytic RNA virus (eg, oncolytic reovirus, oncolytic Newcastle disease virus (NDV), oncolytic measles virus, or oncolytic vesicular stomatitis virus (VSV)).

In some embodiments, the oncolytic virus is a virus, such as a recombinant oncolytic virus, as described in US 2010/0178684 A1, which is incorporated herein by reference in its entirety. In some embodiments, the recombinant oncolytic virus comprises a nucleic acid sequence (eg, a heterologous nucleic acid sequence) encoding an inhibitor of an immune or inflammatory response, for example, as described in US 2010/0178684 A1, which is incorporated herein by reference in its entirety. In an embodiment, a recombinant oncolytic virus, such as an oncolytic NDV, comprises a pro-apoptotic protein (eg, an apoptotic protein), a cytokine (eg, GM-CSF, interferon-gamma, interleukin-2 (IL-2) ), tumor necrosis factor-α), immunoglobulin (example) Such as antibodies against ED-B fibronectin), tumor-associated antigens, bispecific adaptor proteins (eg, bispecific antibodies or antibody fragments directed against NDV HN proteins and T cell co-stimulatory receptors (such as CD3 or CD28); or A fusion protein between human IL-2 and a single chain antibody directed against NDV HN protein). See, for example, Zamarin et al., Future Microbiol. 7.3 (2012): 347-67, which is incorporated herein by reference in its entirety. In some embodiments, the oncolytic virus is a chimeric oncolytic NDV as described in US Pat. No. 8,591,881 B2, US 2012/0122185 A1, or US 2014/0271677 A1, which is incorporated herein by reference in its entirety.

In some embodiments, the oncolytic virus comprises a conditionally replicating adenovirus (CRAd) designed to replicate only in cancer cells. See, for example, Alemany et al. Nature Biotechnol. 18 (2000): 723-27. In some embodiments, the oncolytic adenovirus comprises those described in Table 1 on page 725 of Alemany et al., which is incorporated herein by reference in its entirety.

Exemplary oncolytic viruses include, but are not limited to, the following: Group B oncolytic adenovirus (ColoAd1) (PsiOxus Therapeutics Ltd.) (see, eg, clinical trial identification number: NCT02053220); ONCOS-102 (formerly known as CGTG-102) , which is an adenovirus containing granulocyte-macrophage colony stimulating factor (GM-CSF) (see, eg, clinical trial identification number: NCT01598129); VCN-01, which is genetically encoded by human PH20 hyaluronan Modified oncolytic human adenovirus (VCN Biosciences, SL) (see, eg, clinical trial identification numbers: NCT02045602 and NCT02045589); conditionally replicating adenovirus ICOVIR-5, which is derived from wild-type human adenovirus serotype 5 (Had5) a virus that is modified to selectively replicate in cancer cells with a dysregulated retinoblastoma/E2F pathway (see, eg, clinical trial identification number: NCT01864759); Celyvir, It contains bone marrow-derived autologous mesenchymal stem cells (MSC) infected with ICOVIR5 (an oncolytic adenovirus) (Hospital Infantil Universitario Ni o Jesús, Madrid, Spain/Ramon Alemany) (see eg clinical trial identification number: NCT01844661); CG0070, which is a conditional replication oncolytic serotype 5 adenovirus (Ad5) in which the human E2F-1 promoter drives the basic E1a virus Gene expression, thereby limiting viral replication and cytotoxicity to tumor cells lacking the Rb pathway (Cold Genesys, Inc.) (see eg clinical trial identification number: NCT02143804); or DNX-2401 (formerly known as δ-24-) RGD), an adenovirus that has been engineered to selectively replicate in cells lacking the retinoblastoma (Rb) pathway and more efficiently infect cells expressing certain RGD-binding integrins (Clinica Universidad de Navarra, Universidad De Navarra/DNAtrix, Inc.) (See, for example, Clinical Trial Identification Number: NCT01956734).

In some embodiments, the oncolytic viruses described herein are administered by injection, such as subcutaneous, intraarterial, intravenous, intramuscular, intrathecal or intraperitoneal injection. In embodiments, the oncolytic viruses described herein are administered intratumorally, transdermally, transmucosally, orally, intranasally, or via the lung.

In one embodiment, the CAR-expressing cells described herein are administered to an individual in combination with a molecule that reduces the Treg cell population. Methods for reducing (e.g., depleting) the number of Treg cells are known in the art and include, for example, CD25 depletion, cyclophosphamide administration, modulation of GITR function. Without wishing to be bound by theory, it is desirable to reduce the number of Treg cells in an individual prior to clearance or prior to administration of a CAR-expressing cell as described herein to reduce the number of unnecessary immune cells (eg, Treg) in the tumor microenvironment and reduce the number of individuals. Risk of recurrence. In one embodiment, a CAR-expressing cell described herein is administered in combination with a molecule that targets GITR and/or modulates GITR function, such as a GITR agonist and/or a GITR antibody that depletes regulatory T cells (Treg). individual. In the examples, the CAR-expressing cells described herein are administered to an individual in combination with cyclophosphamide. In one embodiment, the GITR binding molecule and/or modulation is administered prior to administration of the cell expressing the CAR. A molecule that functions as a GITR (eg, a GITR agonist and/or a GITR antibody that depletes Treg). For example, in one embodiment, the GITR agonist can be administered prior to cell clearance. In an embodiment, the cyclophosphamide is administered to the individual prior to administration (eg, infusion or reinfusion) of the cells exhibiting CAR or prior to cell clearance. In an embodiment, the cyclophosphamide and anti-GITR antibody are administered to the individual prior to administration (eg, infusion or reinfusion) of the cells expressing the CAR or prior to cell clearance. In one embodiment, the individual has cancer (eg, a solid cancer or a blood cancer, such as multiple myeloma, ALL or CLL). In one embodiment, the individual has CLL. In an embodiment, the individual has multiple myeloma. In an embodiment, the individual has a solid cancer, such as a solid cancer as described herein. Exemplary GITR agonists include, for example, GITR fusion proteins and anti-GITR antibodies (e.g., bivalent anti-GITR antibodies), such as U.S. Patent No. 6,111,090, European Patent No. 090,505 B1, U.S. Patent No. 8,586,023, PCT Publication No. WO 2010/ GITR fusion protein described in 003118 and WO 2011/090754, or for example, U.S. Patent No. 7,025,962, European Patent No. 1,947,183 B1, U.S. Patent No. 7,812,135, U.S. Patent No. 8,388,967, U.S. Patent No. 8,591,886, Patent No. EP 1866339, PCT Publication No. WO 2011/028683, PCT Publication No. WO 2013/039954, PCT Publication No. WO2005/007190, PCT Publication No. WO 2007/133822, PCT Publication No. WO2005 /055808, PCT Publication No. WO 99/40196, PCT Publication No. WO 2001/03720, PCT Publication No. WO99/20758, PCT Publication No. WO2006/083289, PCT Publication No. WO 2005/115451 Anti-GITR antibodies described in U.S. Patent No. 7,618,632 and PCT Publication No. WO 2011/051726.

In one embodiment, a CAR-expressing cell described herein is administered to an individual in combination with an mTOR inhibitor (eg, an mTOR inhibitor described herein, eg, Repalrog, such as everolimus). In one embodiment, the mTOR inhibitor is administered prior to the cells expressing the CAR. For example, in one embodiment, the mTOR inhibitor can be prior to cell clearance Cast.

In one embodiment, a CAR-expressing cell described herein is administered to an individual in combination with a GITR agonist, such as a GITR agonist described herein. In one embodiment, the GITR agonist is administered prior to the cells expressing the CAR. For example, in one embodiment, the GITR agonist can be administered prior to cell clearance.

In one embodiment, the CAR-expressing cells described herein are administered to a subject in combination with a protein tyrosine phosphatase inhibitor, such as a protein tyrosine phosphatase inhibitor described herein. In one embodiment, the protein tyrosine phosphatase inhibitor is a SHP-1 inhibitor, such as an SHP-1 inhibitor described herein, such as sodium gluconate. In one embodiment, the protein tyrosine phosphatase inhibitor is a SHP-2 inhibitor.

In one embodiment, the CAR-expressing cells described herein can be used in combination with a kinase inhibitor. In one embodiment, the kinase inhibitor is a CDK4 inhibitor, such as a CDK4 inhibitor described herein, such as a CDK4/6 inhibitor, such as 6-ethylindenyl-8-cyclopentyl-5-methyl-2- (5-piperazinyl-1-yl-pyridin-2-ylamino)-8 H -pyrido[2,3- d ]pyrimidin-7-one hydrochloride (also known as palbociclib) Or PD0332991). In one embodiment, the kinase inhibitor is a BTK inhibitor, such as a BTK inhibitor described herein, such as ibrutinib. In one embodiment, the kinase inhibitor is an mTOR inhibitor, such as an mTOR inhibitor described herein, such as rapamycin, a rapamycin analog, OSI-027. The mTOR inhibitor can be, for example, an mTORC1 inhibitor and/or an mTORC2 inhibitor, such as an mTORC1 inhibitor and/or an mTORC2 inhibitor as described herein. In one embodiment, the kinase inhibitor is a MNK inhibitor, such as a MNK inhibitor described herein, such as 4-amino-5-(4-fluoroanilino)-pyrazolo[3,4- d ]pyrimidine . The MNK inhibitor can be, for example, MNK1a, MNK1b, MNK2a, and/or MNK2b inhibitors. In one embodiment, the kinase inhibitor is a dual PI3K/mTOR inhibitor as described herein, such as PF-04695102. In one embodiment, the kinase inhibitor is a DGK inhibitor, such as a DGK inhibitor described herein, such as DGKinh1 (D5919) or DGKinh2 (D5794).

In one embodiment, the kinase inhibitor is a CDK4 inhibitor selected from the group consisting of aloisine A; flavopiridol or HMR-1275, 2-(2-chlorophenyl)-5, 7-Dihydroxy-8-[(3S,4R)-3-hydroxy-1-methyl-4-piperidinyl]-4-chromone; crizotinib (PF-02341066; 2-(2) -chlorophenyl)-5,7-dihydroxy-8-[(2R,3S)-2-(hydroxymethyl)-1-methyl-3-pyrrolidinyl]-4 H -1-benzocytidine Butan-4-one hydrochloride (P276-00); 1-methyl-5-[[2-[5-(trifluoromethyl)-1 H -imidazol-2-yl]-4-pyridyl] oxy] - N - [4- (trifluoromethyl) phenyl] -1 H - benzimidazol-2-amine (of RAF265); edetate Soulain (indisulam) (E7070); Luosiweiting (roscovitine,) (CYC202); palbociclib (PD0332991); dinaciclib (SCH727965); N-[5-[[(5-t-butyloxazol-2-yl)methyl]thio) Thiazol-2-yl]piperidine-4-carboxamide (BMS 387032); 4-[[9-chloro-7-(2,6-difluorophenyl)-5 H -pyrimidine[5,4 - d ][2]Benzazepine-2-yl]amino]-benzoic acid (MLN8054); 5-[3-(4,6-difluoro-1H-benzimidazol-2-yl)-1H -oxazol-5-yl]-N-ethyl-4-methyl-3-pyridylmethylamine (AG-024322); 4-(2,6-dichlorobenzamide ) LH-pyrazole-3-carboxylic acid N- (piperidin-4-yl) Amides (AT7519); 4- [2- methyl-1- (1-methylethyl) -1 H - imidazol - 5- yl] - N - [4- (methyl-sulfo acyl) phenyl] -2-pyrimidinamine (AZD5438); and XL281 (BMS908662).

In one embodiment, the kinase inhibitor is a CDK4 inhibitor, such as Pabbori (PD0332991), and the Pabbori is about 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, 100 mg, 105 mg, 110 mg, 115 mg daily. a dose of 120 mg, 125 mg, 130 mg, 135 mg (eg, 75 mg, 100 mg, or 125 mg) is administered for a period of time, for example, 14 days to 21 days per day for the above-mentioned dose for a period of 28 days or a daily period of 21 days for the above dose. 7 days to 12 days. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles of Pabbori are administered.

In embodiments, a CAR-expressing cell described herein is administered to an individual in combination with a cyclin dependent kinase (CDK) 4 or 6 inhibitor, such as a CDK4 inhibitor or a CDK6 inhibitor as described herein. In the Examples, the CAR-expressing cells and CDK4/6 inhibition described herein Agents (e.g., inhibitors that target CDK4 and CDK6), such as the CDK4/6 inhibitors described herein, are administered to an individual. In one embodiment, the individual has MCL. MCL is an aggressive cancer that responds poorly to currently available therapies, i.e., is substantially incurable. In most cases of MCL, cyclin D1 (a regulator of CDK4/6) is expressed in MCL cells (eg, due to chromosomal translocations involving immunoglobulins and the cyclin D1 gene). Thus, without being bound by theory, it is believed that MCL cells are highly sensitive to CDK4/6 inhibition with high specificity (ie, minimal effect on normal immune cells). CDK4/6 inhibitor alone has some efficacy in the treatment of MCL, but only achieves partial remission and has a high recurrence rate. An exemplary CDK4/6 inhibitor is LEE011 (also known as ribociclib), the structure of which is shown below.

Without being bound by theory, it is believed that the CAR-expressing cells described herein can be administered with a CDK4/6 inhibitor (eg, LEE011 or other CDK4/6 inhibitors described herein) to achieve higher responses, for example, with CDK4/ alone. 6 inhibitors have, for example, a higher rate of remission and/or a lower rate of relapse.

In one embodiment, the kinase inhibitor is a BTK inhibitor selected from the group consisting of: ibrutinib (PCI-32765); GDC-0834; RN-486; CGI-560; CGI-1764; HM-71224; 292; ONO-4059; CNX-774; and LFM-A13. In a preferred embodiment, the BTK inhibitor does not reduce or inhibit the kinase activity of interleukin-2-inducible kinase (ITK) and is selected from the group consisting of GDC-0834; RN-486; CGI-560; CGI-1764 ; HM-71224; CC-292; ONO-4059; CNX-774; and LFM-A13.

In one embodiment, the kinase inhibitor is a BTK inhibitor, such as ibrutinib (PCI-32765). In an embodiment, the CAR-expressing cells described herein are administered to a subject in combination with a BTK inhibitor (eg, ibrutinib). In the examples, the CAR-expressing cells described herein are administered to an individual in combination with ibrutinib (also known as PCI-32765). Structure of Ibrutinib (1-[(3 R )-3-[4-Amino-3-(4-phenoxyphenyl)-1 H -pyrazolo[3,4-d]pyrimidine- 1-Base]piperidin-1-yl]prop-2-en-1-one) is shown below.

In an embodiment, the individual has CLL, mantle cell lymphoma (MCL) or small lymphoblastic lymphoma (SLL). For example, an individual has a deletion in the short arm of chromosome 17 (del(17p), eg in leukemia cells). In other examples, the individual does not have del(17p). In an embodiment, the individual has recurrent CLL or SLL. For example, an individual has previously administered cancer therapy (eg, previously administered one, two, three, or four prior cancer therapies). In an embodiment, the individual has refractory CLL or SLL. In other embodiments, the individual has a follicular lymphoma, such as a relapsed or refractory follicular lymphoma. In some embodiments, Ibrutinib is at about 300-600 mg/day (eg, about 300-350, 350-400, 400-450, 450-500, 500-550, or 550-600 mg/day, eg, about A dose of 420 mg/day or about 560 mg/day, for example, orally. In an embodiment, the ibrutinib is administered at a dose of about 250 mg, 300 mg, 350 mg, 400 mg, 420 mg, 440 mg, 460 mg, 480 mg, 500 mg, 520 mg, 540 mg, 560 mg, 580 mg, 600 mg (eg, 250 mg, 420 mg, or 560 mg) per day. The administration is carried out for a period of time, for example, for a period of 21 days daily with the above dose or for a period of 28 days per day with the above dosage. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles of Ibrutinib are administered. In some embodiments, ibrutinib is administered in combination with rituximab. See, for example, Burger et al. (2013) Ibrutinib In Combination With Rituximab(iR)Is Well Tolerated and Induces a High Rate Of Durable Remissions In Patients With High-Risk Chronic Lymphocytic Leukemia (CLL): New, Updated Results Of a Phase II Trial In 40 Patients, Abstract 675 presented at 55 ^th ASH Annual Meeting and Exposition, New Orleans, LA 7-10 Dec. Without being bound by theory, it is believed that the addition of Ibrutinib enhances the T cell proliferative response and can change T cells from T helper-2 (Th2) to T helper-1 (Th1) phenotype. Th1 and Th2 are phenotypes of helper T cells, in which Th1 contrasts with Th2, guiding different immune response pathways. The Th1 phenotype is associated with a pro-inflammatory response, for example to kill cells, such as intracellular pathogens/viruses or cancer cells, or to persist an autoimmune response. Th2 phenotype is associated with eosinophil accumulation and anti-inflammatory response.

In some embodiments of the methods, uses, and compositions herein, the BTK inhibitor is a BTK inhibitor as described in International Application WO/2015/079417, which is incorporated herein by reference in its entirety. For example, in some embodiments, the BTK inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt thereof;

Wherein R1 is hydrogen, optionally substituted by a hydroxy group, C1-C6 alkyl; R2 is hydrogen or halogen; R3 is hydrogen or halogen; R4 is hydrogen; R5 is hydrogen or halogen; or R4 and R5 are attached to each other and represent Key, -CH2-, -CH2-CH2-, -CH=CH-, -CH=CH-CH2-; -CH2-CH=CH-; or -CH2-CH2-CH2-; R6 and R7 independently of each other represent H, optionally a C1-C6 alkyl group substituted by a hydroxy group, a C3-C6 cycloalkyl group substituted by a halogen or a hydroxy group, or a halogen; R8, R9, R, R', R10 and R11 Independently from each other, H or a C1-C6 alkyl group optionally substituted by a C1-C6 alkoxy group; or both of R8, R9, R, R', R10 and R11 together with the carbon atom to which they are bonded may form a 3-6 membered saturated carbocyclic ring; R12 is hydrogen or optionally substituted by halogen or C1-C6 alkoxy; or R12 and R8, R9, Along with the atoms to which they are combined, a 4, 5, 6 or 7 membered nitrogen heterocycle may be formed which may optionally be substituted by halogen, cyano, hydroxy, C1-C6 alkyl or C1-C6 alkoxy; n is 0 Or 1; and R13 is a C2-C6 alkenyl group optionally substituted by a C1-C6 alkyl group, a C1-C6 alkoxy group or an N,N-di-C1-C6 alkylamino group; optionally a C1-C6 alkane a C2-C6 alkynyl group substituted with a C1-C6 alkoxy group; or a C2-C6 alkylene oxide substituted by a C1-C6 alkyl group as appropriate.

In some embodiments, the BTK inhibitor of Formula I is selected from the group consisting of: N-(3-(5-((1-propenyl)azetidin-3-yl)oxy)-6-aminopyrimidine 4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; (E)-N-(3-(6-amino-5-( (1-(But-2-enyl)azetidin-3-yl)oxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl- 2-fluorobenzamide; N-(3-(6-amino-5-((1-propynyl)azetidin-3-yl)oxy)pyrimidin-4-yl)-5 -fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; N-(3-(6-amino-5-((1-(but-2-ynyl)) Azetidin-3-yl)oxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; N-( 3-(5-((1-Phenylhydrylpiperidin-4-yl)oxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropane N-(3-(6-amino-5-(2-(N-methylpropenyl))ethoxy)pyrimidin-4-yl)-5-fluoro -2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; (E)-N-(3-(6-amino-5-(2-(N-methylbutyl-)- 2-enylamino)ethoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; N-(3-( 6-amino-5-(2-(N-methylpropyne) Amino) ethoxy) pyrimidin-4-yl) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2-fluorobenzonitrile Indoleamine; (E)-N-(3-(6-amino-5-(2-(4-methoxy-N-methylbut-2-enylamino)ethoxy)pyrimidine-4 -yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; N-(3-(6-amino-5-(2-(N-A) Butyl-2-alkynylamino)ethoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; N-( 2-((4-Amino-6-(3-(4-cyclopropyl-2-fluorobenzamide)-5-fluoro-2-methylphenyl)pyrimidin-5-yl)oxy Ethyl)-N-methyloxirane-2-carboxamide; N-(2-((4-amino-6-(3-(6-cyclopropyl-8-fluoro-1-) Side oxyisoquinoline-2(1H)-yl)phenyl)pyrimidin-5-yl)oxy)ethyl)-N-methylpropenylamine; N-(3-(5-(2-propene)醯Aminoethoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; N-(3- (6-Amino-5-(2-(N-ethyl acrylamide)ethoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2 -Fluorobenzamide; N-(3-(6-Amino-5-(2-(N-(2-fluoroethyl) acrylamide)ethoxy)pyrimidin-4-yl)-5- Fluor-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; N-(3-(5-((1-propenylamine)cyclopropyl)methoxy)-6- Aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclo Propyl-2-fluorobenzamide; (S)-N-(3-(5-(2-propenylamine-propoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2 -methylphenyl)-4-cyclopropyl-2-fluorobenzamide; (S)-N-(3-(6-amino-5-(2-(but-2-yneindolyl)) Propyloxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; (S)-N-(3-(6- Amino-5-(2-(N-methylpropenylamine)propoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzene Methionine; (S)-N-(3-(6-Amino-5-(2-(N-methylbut-2-ynindolyl)propoxy)pyrimidin-4-yl)-5 -fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; N-(3-(6-amino-5-(3-(N-methylpropenylamine)) Propoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; (S)-N-(3-(5-( (1-Propylpyridylpyrrolidin-2-yl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluoro Benzalamine; (S)-N-(3-(6-amino-5-((1-(but-2-ynindolyl)pyrrolidin-2-yl)methoxy)pyrimidine-4- 5-(5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; (S)-2-(3-(5-((1-propenylpyridylpyrrolidine) -2-yl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-(hydroxyl) Phenyl)-6-cyclopropyl-3,4-dihydroisoquinolin-1(2H)-one; N-(2-((4-amino-6-(3-(6-cyclopropyl) -1-la-oxy-3,4-dihydroisoquinoline-2(1H)-yl)-5-fluoro-2-(hydroxymethyl)phenyl) Pyrimidin-5-yl)oxy)ethyl)-N-methylpropenylamine; N-(3-(5-((2S,4R)-1-propenyl-4-methoxypyrrolidine) -2-yl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; N-( 3-(6-Amino-5-((2S,4R)-1-(but-2-ynindolyl)-4-methoxypyrrolidin-2-yl)methoxy)pyrimidine-4- 5-(3-(5-((2),4R)-1-propenyl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide 4-methoxypyrrolidin-2-yl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-(hydroxymethyl)phenyl)-6-cyclopropyl- 3,4-dihydroisoquinolin-1(2H)-one; N-(3-(5-((2S,4S)-1-propenyl-4-methoxypyrrolidin-2-yl) Methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; N-(3-(6) -amino-5-(((2S,4S)-1-(but-2-ynindolyl)-4-methoxypyrrolidin-2-yl)methoxy)pyrimidin-4-yl)-5 -fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; N-(3-(5-((2S,4R)-1-propenyl)-4-fluoro Pyrrrolidin-2-yl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; N -(3-(6-Amino-5-((2S,4R)-1-(but-2-yne醯) -4-fluoropyrrolidin-2-yl)methoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; S)-N-(3-(5-((1-propenyl)azetidin-2-yl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2- Methylphenyl)-4-cyclopropyl-2-fluorobenzamide; (S)-N-(3-(6-amino-5-((1-propynyl)-azetidine -2-yl)methoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; (S)-2-(3 -(5-((1-propenylfluorenylazetidin-2-yl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-(hydroxymethyl)phenyl - 6-cyclopropyl-3,4-dihydroisoquinolin-1(2H)-one; (R)-N-(3-(5-((1-propenyl)-azetidine- 2-yl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; (R)- N-(3-(5-((1-propenyl)piperidin-3-yl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)- 4-cyclopropyl-2-fluorobenzamide; N-(3-(5-((2R,3S)-1-propenyl-3-methoxypyrrolidin-2-yl)methoxy) 6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; N-(3-(5-(( (2S,4R)-1-propenyl-4-cyanopyrrolidin-2- Methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; or N-(3-( 5-(((2S,4S)-1-propenyl-4-cyano) Pyrrrolidin-2-yl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide.

Unless otherwise provided, the above chemical terms used to describe the BTK inhibitors of Formula I are used according to the meanings set forth in the International Application No. WO/2015/079417, which is incorporated herein by reference in its entirety.

In one embodiment, the kinase inhibitor is an mTOR inhibitor selected from the group consisting of: temsirolimus; dexamethasone, dimethylphosphinic acid (1 R , 2 R , 4 S ) -4-[(2 R )-2[(1 R ,9 S ,12 S ,15 R ,16 E ,18 R ,19 R ,21 R ,23 S ,24 E ,26 E ,28 Z ,30 S ,32 S ,35 R )-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentamethoxy-11 ,36-dioxa-4-azatricyclo[30.3.1.0 ^4,9 ]hexa(tricadecane)-16,24,26,28-tetraen-12-yl]propyl]-2-methyl Oxycyclohexyl ester, also known as AP23573 and MK8669; everolimus (RAD001); rapamycin (AY22989); simapiride; (5-{2,4-bis[(3S)-3- Methylmorpholin-4-yl]pyrido[2,3- d ]pyrimidin-7-yl}-2-methoxyphenyl)methanol (AZD8055); 2-amino-8-[anti-4- (2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridyl)-4-methyl-pyrido[2,3-d]pyrimidin-7(8 H )-one (PF04691502); and N ² -[1,4-di- oxy-4-[[4-(4-trioxy-8-phenyl- 4H- 1-benzopipene-2-yl) Morpholinium-4-yl]methoxy]butyl]-L-spermine guanylglycine decyl-L-α-aspartame decyl L-serine-(SEQ ID NO: 383), Inner salt (SF1126); and XL765

In one embodiment, the kinase inhibitor is an mTOR inhibitor, such as rapamycin, and the rapamycin is administered at a dose of about 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg (eg, 6 mg) per day. The administration is carried out for a period of time, for example, for a period of 21 days daily with the above dose or for a period of 28 days per day with the above dosage. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles of rapamycin are administered. In one embodiment, the kinase inhibitor is an mTOR inhibitor, such as everolimus, and everolimus is about 2 mg, 2.5 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg per day, 11mg, 12mg, 13mg, 14mg, 15mg The dose (e.g., 10 mg) is administered for a period of time, such as a daily period of 28 days for the above dose. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles of everolimus are administered.

In one embodiment, the kinase inhibitor is a MNK inhibitor selected from the group consisting of CGP052088; 4-amino-3-(p-fluoroanilino)-pyrazolo[3,4- d ]pyrimidine (CGP57380); Cercosporamide; ETC-1780445-2; and 4-amino-5-(4-fluoroanilino)-pyrazolo[3,4-d]pyrimidine.

In an embodiment, a CAR-expressing cell described herein is associated with a phosphoinositide 3-kinase (PI3K) inhibitor (eg, a PI3K inhibitor as described herein, eg, idelalisib or duvelisib) And/or rituximab in combination with the individual. In the Examples, the CAR-expressing cells described herein are administered to an individual in combination with Adesb and Rituximab. In the Examples, the CAR-expressing cells described herein are administered to a subject in combination with Duvis and rituximab. Edsb (also known as GS-1101 or CAL-101; Gilead) is a small molecule that blocks the δ isoform of PI3K. Structure of Adesb (5-fluoro-3-phenyl-2-[(1S)-1-(7 H -嘌呤-6-ylamino)propyl]-4(3 H )-quinazoline Ketones are shown below.

Duwei Lisi (also known as IPI-145; Infinity Pharmaceuticals and Abbvie) is a small molecule that blocks PI3K-δ, γ. The structure of Duwei Lisi (8-chloro-2-phenyl-3-[(1S)-1-(9H-嘌呤-6-ylamino)ethyl]-1(2H)-isoquinolinone) In the following.

In an embodiment, the individual has CLL. In an embodiment, the individual has recurrent CLL, eg, the individual has previously been administered a cancer therapy (eg, previously administered anti-CD20 antibody or previously administered ibrutinib). For example, an individual has a deletion in the short arm of chromosome 17 (del(17p), eg in leukemia cells). In other examples, the individual does not have del(17p). In an embodiment, the individual comprises a leukemia cell comprising a mutation in an immunoglobulin heavy chain variable region ( IgV _H ) gene. In other embodiments, the individual does not comprise leukemia cells comprising a mutation in the immunoglobulin heavy chain variable region ( IgV _H ) gene. In an embodiment, the individual has a deletion (del(11q)) in the long arm of chromosome 11. In other examples, the individual does not have del(11q). In an embodiment, Adesbu is about 100-400 mg (eg, 100-125, 125-150, 150-175, 175-200, 200-225, 225-250, 250-275, 275-300, 325- A dose of 350, 350-375 or 375-400), such as BID administration. In an embodiment, the Duvis is administered at a dose of about 15-100 mg (e.g., about 15-25, 25-50, 50-75, or 75-100 mg), for example, twice a day. In an embodiment, rituximab is at a dose of about 350-550 mg/m ² (eg, 350-375, 375-400, 400-425, 425-450, 450-475, or 475-500 mg/m ² ), For example, intravenous administration.

In the Examples, the CAR-expressing cells described herein are administered to an individual in combination with a pleomorphic lymphoma kinase (ALK) inhibitor. Exemplary ALK kinases include, but are not limited to, Pfizer, Novartis, alectinib (Chugai), Buginitinib (also known as AP26113; Ariad) ), entrectinib (Ignyta), PF-06463922 (Pfizer), TSR-011 (Tesaro) (see, for example, Clinical Trial Identification No. NCT02048488), CEP-37440 (Teva), and X-396 (Xcovery). In some embodiments, the individual has a solid cancer, such as described herein Solid cancer, such as lung cancer.

The chemical name of klotinib is 3-[(1 R )-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-5-(1-piperidin-4-ylpyrazole- 4-yl)pyridin-2-amine. The chemical name of ceritinib is 5-chloro- N ² -[2-isopropoxy-5-methyl-4-(4-piperidinyl)phenyl]- N ⁴ -[2-(isopropyl Alkylsulfonyl)phenyl]-2,4-pyrimidinediamine. The chemical name of erlotinib is 9-ethyl-6,6-dimethyl-8-(4-morpholinylpiperidin-1-yl)-11- oxo-6,11-dihydro- 5H-benzo[b]carbazole-3-carbonitrile. The chemical name of buginib is 5-chloro-N ² -{4-[4-(dimethylamino)-1-piperidinyl]-2-methoxyphenyl}-N ⁴ -[2 -(Dimethylphosphonium)phenyl]-2,4-pyrimidinediamine. The chemical name of entrinib is N-(5-(3,5-difluorobenzyl)-1H-indazol-3-yl)-4-(4-methylpiperazin-1-yl)- 2-((Tetrahydro-2H-piperidin-4-yl)amino)benzamide. The chemical name of PF-06463922 is (10R)-7-amino-12-fluoro-2,10,16-trimethyl-15-sideoxy-10,15,16,17-tetrahydro-2H-8 4-(methylene bridge) pyrazolo[4,3-h][2,5,11]-benzoxazacyclotetradecane-3-carbonitrile. The chemical name of CEP-37440 is (S)-2-((5-chloro-2-((6-(4-(2-hydroxyethyl))piperazin-1-yl)-1-methoxy-6) , 7,8,9-Tetrahydro-5H-benzo[7] ran-2-yl)amino)pyrimidin-4-yl)amino)-N-methylbenzamide. The chemical name of X-396 is (R)-6-amino-5-(1-(2,6-dichloro-3-fluorophenyl)ethoxy)-N-(4-(4-methyl) Piperazine-1-carbonyl)phenyl)pyridazine-3-carboxamide.

In one embodiment, the kinase inhibitor is a dual phospholipid inositol 3-kinase (PI3K) and mTOR inhibitor selected from the group consisting of 2-amino-8-[trans-4-(2-hydroxyethoxy) Cyclohexyl]-6-(6-methoxy-3-pyridyl)-4-methyl-pyrido[2,3- d ]pyrimidin-7(8 H )-one (PF-04691502); N - [4-[[4-(Dimethylamino)-1-piperidinyl]carbonyl]phenyl] -N '-[4-(4,6-di-4-morpholinyl-1,3, 5-triazin-2-yl)phenyl]urea (PF-05212384, PKI-587); 2-methyl-2-{4-[3-methyl-2-oxo-8-(quinoline) 3-yl)-2,3-dihydro-1 H -imidazo[4,5-c]quinolin-1-yl]phenyl}propionitrile (BEZ-235); apitolisib (GDC-0980, RG7422); 2,4-difluoro-N-{2-(methyloxy)-5-[4-(4-pyridazinyl)-6-quinolinyl]-3-pyridine Benzosulfonamide (GSK2126458); 8-(6-methoxypyridin-3-yl)-3-methyl-1-(4-(piperazin-1-yl)-3-(trifluoromethyl) Phenyl)-1H-imidazo[4,5-c]quinoline-2(3H)-one maleic acid (NVP-BGT226); 3-[4-(4-morpholinylpyridine) [3',2':4,5]furo[3,2-d]pyrimidin-2-yl]phenol (PI-103); 5-(9-isopropyl-8-methyl-2-( N-morpholinyl)-9H-indol-6-ylpyrimidin-2-amine (VS-5584, SB2343); and N-[2-[(3,5-di) Methoxyphenyl) amino] quinoxalin-3-yl] -4 - [(4-methyl-3-methoxyphenyl) carbonyl] aminophenyl sulfonamide Amides (XL765).

A drug that inhibits the calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or inhibits p70S6 kinase (rapamycin), which is important for growth factor-induced signaling, may also be used (Liu et al., Cell 66: 807-815, 1991; Henderson et al, Immun. 73: 316-321, 1991; Bierer et al, Curr. Opin. Immun. 5: 763-773, 1993). In another aspect, the cellular composition of the invention can be administered to a patient (eg, before, simultaneously, or after) in combination with: bone marrow transplantation, T cell ablation therapy using a chemotherapeutic agent such as fludarabine, external beam Radiation therapy (XRT), cyclophosphamide and/or antibodies such as OKT3 or CAMPATH. In one aspect, the cellular composition of the invention is administered following B cell ablation therapy, such as an agent that reacts with CD20, such as Rituxan. For example, in one embodiment, an individual may be subjected to standard treatment using high dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, the individual receives the expanded immune cell infusion of the invention after transplantation. In another embodiment, the expanded cells are administered before or after surgery.

In one embodiment, the CAR-expressing cells described herein are administered to a subject in combination with a bisphosphonate such as Pamidronate (Aredia®); Zoledronic acid or azole. Zoledronate (Zometa®, Zomera®, Aclasta® or Reclast®); Alendronate (Fosamax®); Risedronate (Actonel®); Ibandron Ibandronate (Boniva®); Clodrodronate (Bonefos®); Etidronate (Didronel®); Tiludronate (Skelid®); Pami Pholide (Aredia®); Neridronate (Nerixia®); strontium ranelate (Protelos® or Protos®); and Teriparatide (Forteo®).

In one embodiment, the CAR-expressing cells described herein are administered to a subject in combination with a corticosteroid such as dexamethasone (eg, Decadron®), beclomethasone (eg, Beclovent®), cortisol (also Known as cortisone, sodium cortisol succinate, sodium hydroxycorticosterone and sold under the trade names Ala-Cort®, hydroxycorticostere phosphate, Solu-Cortef®, Hydrocort Acetate® and Lanacort®, and nylon (sold under the trade names δ-Cortel®, Orapred®, Pediapre® and Prelene®), prednisone (sold under the trade names Deltasoone®, LiquidRed®, Meticorten® and Orasone®), methylprednisolone (also known as 6) - methylprednisolone, methylprednisolone acetate, methylprednisolone sodium succinate, sold under the trade names Duralone®, Medralone®, Medrol®, M-Prednisol® and Solu-Medrol®; antihistamines, Such as diphenhydramine (such as Benadryl®), hydroxyzine and cyproheptadine; and bronchodilators, such as beta-adrenergic receptor agonists, albuterol (eg Proventil®) and special He Lin (terbutaline) (Brethine®).

In one embodiment, the CAR-expressing cells described herein are administered to an individual in combination with an immunomodulatory agent such as aftopuzumab (available from Roche®); Nephilsta®; Nalamine (CC-5013, Revlimid®); Thalamid®; Actimid (CC4047); and IRX-2 (including interleukin 1, interleukin 2 and interferon) A mixture of gamma human cytokines, CAS 951209-71-5, available from IRX Therapeutics).

In one embodiment, the CAR-expressing cells described herein are administered to a subject in combination with a proteasome inhibitor such as bortezomib (Velcade®); Ixazomib citrate (MLN9708, CAS 1201902) -80-8); Danoprevir (RG7227, CAS 850876-88-9); Isazomi (MLN2238, CAS 1072833-77-2); and (S)-N-[(phenyl Methoxy)carbonyl]-L-alkamine-N- (1-Minyl-3-methylbutyl)-L-alkamine (MG-132, CAS 133407-82-6).

In one embodiment, the CAR-expressing cells described herein are administered to a subject in combination with a vascular endothelial growth factor (VEGF) receptor such as bevacizumab (Avastin®) or axitinib (Inlyta®). Brinitib alaninate (BMS-582664, 2-aminoaminopropionic acid ( S )-(( R )-1-(4-(4-fluoro-2-methyl-) 1 H -indol-5-yloxy)-5-methylpyrrolo[2,1- f ][1,2,4]triazin-6-yloxy)propan-2-yl) Sorafenib (Nexavar®); Pazopanib (Votrient®); Sunitinib malate (Sutent®); Cediranib (AZD2171, CAS 288383-20-1); Vargatef (BIBF1120, CAS 928326-83-4); Foretinib (GSK1363089); Telatinib (BAY57-9352, CAS 332012-40-5); Apatinib (YN968D1, CAS 811803-05-1); Imatinib (Gleevec®); Ponatinib (AP24534, CAS 943319-70) -8); Tivozanib (AV951, CAS 475108-18-0); Regorafenib (BAY73-4506, CAS 755037-03-7); Vanaline dihydrochloride (PT K787, CAS 212141-51-0); Brivinib (BMS-540215, CAS 649735-46-6); Vandetanib (Caprelsa® or AZD6474); Mordisani diphosphate Motesanib diphosphate (AMG706, CAS 857876-30-3, N-(2,3-dihydro-3,3-dimethyl-1H-indol-6-yl)-2-[(4-pyridine) Methyl)amino]-3-pyridinecarboxamide, described in PCT Publication No. WO 02/066470); Dovitinib dilactic acid (TKI258, CAS 852433-84-2) ; Linfanib (ABT869, CAS 796967-16-3); Cabozinitinib (XL184, CAS 849217-68-1); Lasturtinib (CAS 111358-88- 4); N-[5-[[[5-(1,1-dimethylethyl)-2-oxazolyl]methyl]thio]-2-thiazolyl]-4-piperidinecarboxamidine Amine (BMS38703, CAS 345627-80-7); (3R,4R)-4-amino-1-((4-((3-methoxyphenyl)amino)pyrrolo[2,1-f ][1,2,4]triazin-5-yl)methyl)piperidin-3-ol (BMS690514); N- (3,4-dichloro-2-fluorophenyl)-6-methoxy -7-[[(3aα,5β,6aα)-octahydro-2-methylcyclopenta[ c ]pyrrole-5-yl]methoxy]-4-quinazolinamine (XL647, CAS 781613-23 -8); 4-methyl-3-[[1-methyl-6-(3-pyridine) Yl) -1 H - pyrazolo [3,4- d] pyrimidin-4-yl] amino] - N - [3- (trifluoromethyl) phenyl] - benzoyl amine (BHG712, CAS 940310 -85-0); and Aflibercept (Eylea®).

In one embodiment, the CAR-expressing cells described herein are administered to a subject in combination with a CD20 antibody or a combination thereof, such as rituximab (Riuxan® and MabThera®); and tosimozumab (Bexxar®) And; atorumumab (Arzerra®), bubrimumab tiuxetan (Zevalin®); and tocilizumab.

In one embodiment, the CAR-expressing cells described herein are administered to an individual in combination with an anticonvulsant, such as an anticonvulsant (anti-epileptic or anti-epileptic drug): an aldehyde, such as paraldehyde; an aromatic allylate Alcohols such as stiripentol (Diacomit®); barbiturates such as phenobarbital (Luminal®), mebaral®, barbexaclone (Maliasin®); benzodiazepines, such as clobazam (Onfi®), clonazepam (Klonopin®), clorazepate (Tranxene® and Novo-) Clopate®), diazepam (Valium®, Lembrol®, Diastat®), midazolam (Versed®), lorazepam (Ativan® and Orfidal®), nitrazepam ( Nitrazepam) (Alodorm®, Arem®, Insoma®), temazepam (restoril®, Normison®), nimetzepam (Erimin®); bromide such as potassium bromide; uric acid Esters, such as felbamate (Felbatol®); formamide, such as carbamazepine (Tegretol®, Equetro®), oxcarbaze (oxcarbaze) Pine) (Trileptal®, Oxcarb®), eslicarbazepine acetate (Aptiom®); fatty acids such as valproate (valproic acid, sodium valproate, sodium divalproate), hi Vigabatrin (Sabril®), Progabide (Gabren®), tiagabine (Gabitril®); fructose derivatives such as topiramate (Topamax®); GABA analogues such as gabapentin (Neurontin®), Pregabalin (Lyrica®); carbendazim, such as ethotoin (Peganone®), phenytoin (Dilantin®), mephenytoin (Mesantoin®), phenytoin (fosphenytoin) (Cerebyx®, Prodilantin®); oxazolidinedione, such as paramethadione (Paradione®), trimethadione (Tridione®); propionate, such as benzprofen (beclamide) (Choracon®, Hibicon®, Posedrine®); pyrimidinedione, such as primidone (Mysoline®); pyrrolidine, such as bovisracetan (brivaracetam), levetiracetam (levetiracetam) ), Seletracetam (Keppra®); Dibutylimine, such as ethosuximide (Zarontin®), phensuximide (Milontin®), mesalimide (mesuximide) Celontin®, Petinutin®); sulfonamides such as acetazolamide (Diamox®), Shu Sultiame (Ospolot®), methazolamide (Neptazane®), zonisamide (Zonegran®); triazines such as lamotrigine (Lamictal®); Urea, such as pheneturide, phenacemide (Phenurone®); pentamidine (proline derivative of valproate), such as valpromide (Depamide®) ), valnoctamide; an AMPA receptor antagonist, such as perampanel (Fycompa®).

In the Examples, the CAR-expressing cells described herein are administered to an individual in combination with a guanamine 2,3-dioxygenase (IDO) inhibitor. IDO is an enzyme that catalyzes the degradation of the amino acid L-tryptophan to kynurenine. Many cancers overexpress IDO, such as prostate cancer, colorectal cancer, pancreatic cancer, cervical cancer, stomach cancer, ovarian cancer, head cancer, and lung cancer. pDC, macrophages, and dendritic cells (DC) can express IDO. Without being bound by theory, it is believed that L-tryptophan is reduced (eg, catalyzed by IDO) to cause an immunosuppressive ring by inducing T cell disability and apoptosis. territory. Thus, without being bound by theory, it is believed that IDO inhibitors can enhance the efficacy of the CAR-expressing cells described herein, for example, by reducing the suppression or death of immune cells that express CAR. In an embodiment, the individual has a solid tumor, such as a solid tumor as described herein, such as prostate cancer, colorectal cancer, pancreatic cancer, cervical cancer, gastric cancer, ovarian cancer, head cancer, or lung cancer. Exemplary IDO inhibitors include, but are not limited to, 1-methyl-tryptophan, indoximod (NewLink Genetics) (see, eg, clinical trial identification number NCT01191216; NCT01792050) and INCB024360 (Incyte Corp.) ( See, for example, clinical trial identification number NCT01604889; NCT01685255).

In the Examples, the CAR-expressing cells described herein are administered to a subject in combination with a modulator of bone marrow-derived suppressor cells (MDSC). MDSC accumulates in the peripheral and many tumor sites of solid tumors. These cells inhibit the T cell response, thereby impeding the efficacy of cell therapy that expresses CAR. Without being bound by theory, it is believed that administration of an MDSC modulator enhances the efficacy of the cells of the CAR described herein. In one embodiment, the individual has a solid tumor, such as a solid tumor described herein, such as a glioblastoma. Exemplary MDSC modulators include, but are not limited to, MCS110 and BLZ945. MCS110 is a monoclonal antibody (mAb) directed against macrophage community stimulating factor (M-CSF). See, for example, clinical trial identification number NCT00757757. BLZ945 is a small molecule inhibitor of the Community Stimulating Factor 1 Receptor (CSF1R). See, for example, Pyonteck et al. Nat. Med. 19 (2013): 1264-72. The structure of BLZ945 is shown below.

In the Examples, the CAR-expressing cells described herein are administered to an individual in combination with a CD19 CART cell (eg, CTL 019, eg, as described in WO 2012/079000, herein incorporated by reference). In an embodiment, the individual has acute myeloid leukemia (AML), such as CD19 positive AML or CD19 negative AML. In an embodiment, the individual has a CD19+ lymphoma, such as CD19+ non-Hodgkin's lymphoma (NHL), CD19+ FL, or CD19+ DLBCL. In an embodiment, the individual has a relapsed or refractory CD19+ lymphoma. In an embodiment, lymphocyte depletion chemotherapy is administered to an individual prior to, concurrently with, or subsequent to administration (eg, infusion) of the CD19 CART cells. In one example, lymphocyte depletion chemotherapy is administered to an individual prior to administration to CD19 CART cells. For example, lymphocyte depletion chemotherapy ends 1-4 days (eg, 1, 2, 3, or 4 days) prior to infusion of CD19 CART cells. In an embodiment, multiple doses of CD19 CART cells are administered, for example, as described herein. For example, a single dose contains about 5 x 10 ⁸ CD19 CART cells. In an embodiment, lymphocyte depletion chemotherapy is administered to a subject prior to, concurrently with, or subsequent to administration of (eg, infusion) a cell expressing CAR as described herein, eg, a cell that exhibits non-CD19 CAR. In an embodiment, the CD19 CART is administered to a subject prior to, concurrently with, or subsequent to administration of a cell that exhibits non-CD19 CAR, such as a cell that exhibits non-CD19 CAR as described herein.

In some embodiments, a CAR-expressing cell described herein is administered to a subject in combination with a cell that exhibits CD19 CAR (eg, CTL 019, eg, as described in WO 2012/079000, herein incorporated by reference) for BCMA exhibits a disease associated with it, such as the cancer described herein. Without being bound by theory, the combination of cells expressing CD19 CAR and cells expressing CAR can regulate immune responses, deplete B cells, and/or improve the tumor microenvironment by targeting early lineage cancer cells, such as cancer stem cells. To enhance the efficacy of the cells expressing CAR as described herein. For example, cells expressing CD19 CAR target cancer cells that exhibit early lineage markers, such as cancer stem cells and cells expressing CD19, while the cells expressing CAR described herein are targeted to express late lineage markers, such as BCMA cancer. cell. This pre-adjustment method enhances the efficacy of the cells expressing CAR as described herein. In such embodiments, the cells expressing CD19 CAR are administered prior to, concurrently with, or subsequent to administration (e.g., infusion) of the cells of the CAR described herein.

In the examples, the cells expressing CAR described herein also exhibit targeting to CD19. CAR, such as CD19 CAR. In one embodiment, the CAR-expressing cells and CD19 CAR described herein are administered to an individual to treat a cancer described herein, such as AML. In one embodiment, the configuration of one or both of the CAR molecules comprises a primary intracellular signaling domain and a costimulatory signaling domain. In another embodiment, the configuration of one or both of the CAR molecules comprises a primary intracellular signaling domain and two or more, eg, 2, 3, 4 or 5 or more co-stimulatory signals Conduction domain. In such embodiments, the CAR molecules and CD19 CARs described herein may have the same or different major intracellular signaling domains, the same or different co-stimulatory signaling domains, or the same number or a different number of co-stimulatory signaling structures. area. Alternatively, the CAR and CD19 CAR described herein are configured to split CAR, wherein one of the CAR molecules comprises an antigen binding domain and a co-stimulatory domain (eg, 4-1BB), while another CAR molecule comprises an antigen binding domain and A major intracellular signaling domain (eg, CD3ξ).

In some embodiments, the CAR-expressing cell and interleukin-15 (IL-15) polypeptide, interleukin-15 receptor alpha (IL-15Ra) polypeptide or IL-15 polypeptide and IL-15Ra are described herein. A combination of both polypeptides (eg, hetIL-15 (Admune Therapeutics, LLC)) is administered in combination to the individual. hetIL-15 is a heterodimeric non-covalent complex of IL-15 and IL-15Ra. The hetIL-15 is described, for example, in U.S. Patent No. 8,124,084, U.S. 2012/0177598, U.S. 2009/0082299, U.S. 2012/0141413, and U.S. 2011/0081311. In the examples, het-IL-15 was administered subcutaneously. In an embodiment, the individual has a cancer, such as a solid cancer, such as melanoma or colon cancer. In an embodiment, the individual has a metastatic cancer.

In one embodiment, an agent that reduces or ameliorates the side effects associated with administration of cells expressing CAR can be administered to an individual. Side effects associated with administration of cells expressing CAR include, but are not limited to, CRS and hemophagocytic lymphohistiocytosis (HLH), also known as macrophage activation syndrome (MAS). Symptoms of CRS include high fever, nausea, transient hypotension, hypoxia, and the like. CRS can include clinical signs and symptoms, such as fever, fatigue Lack, loss of appetite, muscle pain, joint pain, nausea, vomiting and headache. CRS can include clinical signs and symptoms of the skin, such as a rash. CRS can include clinical signs and symptoms of the stomach, such as nausea, vomiting, and diarrhea. CRS can include clinical signs and symptoms of the respiratory tract, such as shortness of breath and hypoxemia. CRS can include clinical cardiovascular signs and symptoms such as tachycardia, widened pulse pressure, hypotension, increased cardiac output (early), and potentially reduced cardiac output (late). The CRS can include clinical signs and symptoms of hypertension, such as elevated d-dimers, fibrinogenemia with or without bleeding. CRS can include clinical signs and symptoms of the kidney, such as nitrogenemia. CRS can include clinical liver signs and symptoms such as elevated transaminase and hyperbilirubinemia. CRS can include clinical signs and symptoms such as headache, mental state changes, confusion, convulsions, difficulty in calling words or Frank aphasia, hallucinations, tremors, dymetria, gait changes, and epilepsy.

Thus, the methods described herein can comprise administering to a subject a cell expressing CAR as described herein and further administering one or more agents that increase the level of soluble factor, which is caused by treatment with a cell that exhibits CAR. In one embodiment, the elevated soluble factor in the individual is one or more of IFN-[gamma], TNF[alpha], IL-2, and IL-6. In one embodiment, the elevated factor in the individual is one or more of IL-1, GM-CSF, IL-10, IL-8, IL-5, and fraktalkine. Thus, an agent administered to treat such side effects can be an agent that counteracts one or more of these soluble factors. In one embodiment, the agent that counteracts one or more of these soluble forms is an antibody or antibody fragment. Examples of such agents include, but are not limited to, steroids (eg, corticosteroids), TNFα inhibitors, and IL-6 inhibitors. An example of a TNFα inhibitor is an anti-TNFα antibody molecule, such as infliximab, adalimumab, certolizumab pegol, and golimumab (golimumab) ). Another example of a TNFα inhibitor is a fusion protein, such as entanercept. Small molecule inhibitors of TNFα include, but are not limited to, xanthine derivatives (such as pentoxifylline) and bupropion. An example of an IL-6 inhibitor is an anti-IL-6 antibody molecule, such as tocilizumab (tocilizumab) (toc), sarilumab (sarilumab), esilimomab, CNTO 328, ALD518/BMS-945429, CNTO 136, CPSI-2364, CDP6038, VX30, ARGX-109, FE301 And FM101. In one embodiment, the anti-IL-6 antibody molecule is tocilizumab. An example of an inhibitor based on IL-1R is anakinra.

In some embodiments, the subject is administered a corticosteroid, such as, for example, methylprednisolone, hydrocorticosterone.

In some embodiments, the vasopressor is administered to the individual, such as norepinephrine, dopamine, phenylephrine, epinephrine, vasopressin, or a combination thereof.

In an embodiment, the antipyretic can be administered to the individual. In one embodiment, an analgesic can be administered to an individual.

In one embodiment, the individual can be administered an agent that prevents the cells expressing BCMA CAR from being transported to the brain, such as natalizumab (TYSABRI®). BCMA expression has been detected in one part of the brain, such as the cerebellum or medulla, such as its splice variant. Without being bound by any particular theory, preventing the transport of cells expressing BCMA CAR to the brain preferably prevents any cells expressing BCMA CAR from interacting with or acting on brain tissue that exhibits BCMA.

In one embodiment, an agent that increases the activity of a cell expressing CAR can be administered to an individual. For example, in one embodiment, the agent can be an agent that inhibits the inhibitory molecule, for example, the agent is a checkpoint inhibitor. In some embodiments, an inhibitory molecule (eg, stylized death 1 (PD1)) can reduce the ability of a cell expressing CAR to establish an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (eg, CEACAM-1, CEACAM-3, and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine and TGFR beta. For example by Inhibition of DNA, RNA or protein levels to inhibit inhibitory molecules optimizes the performance of cells expressing CAR. In an embodiment, for example, as described herein, a nucleic acid, such as an inhibitory nucleic acid, such as a dsRNA, such as an siRNA or shRNA, is clustered in a regularly spaced short palindromic repeat (CRISPR), a transcriptional activator-like effector nuclease (TALEN) or Zinc finger endonuclease (ZFN) can be used to inhibit the expression of inhibitory molecules in cells expressing CAR. In one embodiment, the inhibitor is shRNA. In one embodiment, the inhibitory molecules in the cells expressing CAR are inhibited. In such embodiments, the dsRNA molecule that inhibits the expression of the inhibitory molecule is linked to a nucleic acid encoding a component of the CAR (eg, all components). In an embodiment, the CAR-expressing cells described herein are administered in combination with an inhibitor of an inhibitory molecule, such as a checkpoint inhibitor, for example, in combination with an inhibitor of PD1 and/or PD-L1. In the Examples, the CAR-expressing cells described herein are administered in combination with a PD1 inhibitor. In the examples, the CAR-expressing cells described herein are administered in combination with a PD-L1 inhibitor.

In one embodiment, a nucleic acid molecule encoding a dsRNA molecule that inhibits expression of a molecule that modulates or modulates (eg, inhibits) T cell function is operably linked to a promoter, eg, a promoter derived from H1 or U6, such that inhibition of regulation or The expression of a dsRNA molecule that modulates (eg, inhibits) the expression of a molecule of T cell function, for example, is manifested in a cell that exhibits CAR. See, for example, Tiscornia G., "Development of Lentiviral Vectors Expressing siRNA", Chapter 3, Gene Transfer: Delivery and Expression of DNA and RNA (edited by Friedmann and Rossi). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2007; Brummelkamp TR et al, (2002) Science 296: 550-553; Miyagishi M et al, (2002) Nat. Biotechnol. 19: 497-500. In one embodiment, a nucleic acid molecule encoding a dsRNA molecule that inhibits expression of a molecule that modulates or modulates (eg, inhibits) T cell function is present on the same vector comprising a nucleic acid molecule encoding a component of the CAR (eg, all components) (eg, On the lentiviral vector). In such embodiments, a nucleic acid molecule encoding a dsRNA molecule that inhibits expression of a molecule that modulates or modulates (eg, inhibits) T cell function is located on a vector (eg, a lentiviral vector) encoding a component of the CAR (eg, all components) 5' or 3' of the nucleic acid. A nucleic acid molecule encoding a dsRNA molecule that inhibits expression of a molecule that modulates or modulates (eg, inhibits) T cell function can be transcribed in the same or a different direction than the nucleic acid encoding a component of the CAR (eg, all components). In one embodiment, a nucleic acid molecule encoding a dsRNA molecule that inhibits expression of a molecule that regulates or modulates (eg, inhibits) T cell function is present in addition to a vector comprising a nucleic acid encoding a component of the CAR (eg, all components) In one embodiment, a nucleic acid molecule encoding a dsRNA molecule that inhibits expression of a molecule that modulates or modulates (eg, inhibits) T cell function is transiently expressed in a cell that exhibits CAR. In one embodiment, the coding inhibition is regulated or A nucleic acid molecule of a dsRNA molecule that modulates (eg, inhibits) the expression of a molecule of T cell function is stably integrated into the genome of a cell expressing CAR. Figures 41A-41E depict vectors for representing components of CAR (eg, all components). An example of a dsRNA molecule that inhibits the expression of a molecule that modulates or modulates (eg, inhibits) T cell function.

Examples of dsRNA molecules suitable for inhibiting the expression of molecules that regulate or modulate (e.g., inhibit) T cell function are provided below, wherein the molecule that modulates or modulates (e.g., inhibits) T cell function is PD-1.

The name of the PDCD1 (PD1) RNAi agent (derived from its position in the mouse PDCD1 gene sequence NM_008798.2) and SEQ ID NO: 286-333 representing the DNA sequence are provided in Table 18 below. Both sense (S) and antisense (AS) sequences are presented in this table as 19-mer and 21-mer sequences. It should also be noted that the position (PoS, eg, 176) is derived from the position number in the mouse PDCD1 gene sequence NM_008798.2. SEQ ID NO corresponds to "Sense 19" SEQ ID NO: 286-297; "Sense 21" SEQ ID NO: 298-309; "Antisense 21" SEQ ID NO: 310-321; "Antisense 19" Indicated in the 12 groups of SEQ ID NOS: 322-333.

The name of the PDCD1 (PD1) RNAi agent (derived from its position in the human PDCD1 gene sequence) and SEQ ID NO. 334-381 representing the DNA sequence are provided in Table 19 below. The sense (S) and antisense (AS) sequences are presented as 19-mer and 21-mer sequences. SEQ ID NO corresponds to "Sense 19" SEQ ID NO: 334-345; "Sense 21" SEQ ID NO: 346-357; "Antisense 21" SEQ ID NO: 358-369; "Antisense 19" Indicated in the 12 groups of SEQ ID NOs: 370-381.

In one embodiment, the inhibitor of the inhibitory signal can be, for example, an antibody or antibody fragment that binds to the inhibitory molecule. For example, the agent can be an antibody or antibody fragment that binds to PD1, PD-L1, PD-L2, or CTLA4 (eg, ipilimumab (also known as MDX-010 and MDX-101, and Yervoy) ® sold; Bristol-Myers Squibb); Tremelimumab (IgG2 monoclonal antibody, obtained from Pfizer, formerly known as ticilimumab, CP-675, 206)). In one embodiment, the agent is an antibody or antibody fragment that binds to TIM3. In one embodiment, the agent is an antibody or antibody fragment that binds to LAG3. In an embodiment, an agent that enhances the activity of a cell that exhibits CAR, such as an inhibitor of an inhibitory molecule, is administered in combination with an allogeneic CAR, such as the allogeneic CAR described herein (eg, as described in the Allogeneic CAR section herein). versus.

PD-1 is an inhibitory member of the CD28 receptor family, which also includes CD28, CTLA-4, ICOS, and BTLA. PD-1 is expressed on activated B cells, T cells, and bone marrow cells (Agata et al. 1996 Int. Immunol 8: 765-75). Two ligands PD-1 of PD-1 And PD-L2 have been shown to downregulate T cell activation upon binding to PD-1 (Freeman et al, 2000 J Exp Med 192: 1027-34; Latchman et al. 2001 Nat Immunol 2: 261-8; Carter et al. 2002 Eur J Immunol 32:634-43). PD-L1 is abundantly present in human cancers (Dong et al. 2003 J Mol Med 81: 281-7; Blank et al. 2005 Cancer Immunol. Immunother 54: 307-314; Konishi et al. 2004 Clin Cancer Res 10: 5094). Immunosuppression can be reversed by inhibiting the local interaction of PD-1 with PD-L1. Antibodies, antibody fragments and other inhibitors of PD-1, PD-L1 and PD-L2 are available in the art and can be used in combination with the CAR of the invention described herein. For example, nivolumab (also known as BMS-936558 or MDX1106; Bristol-Myers Squibb) is a fully human IgG4 monoclonal antibody that specifically blocks PD-1. Niprozumab (pure line 5C4) and other human monoclonal antibodies that specifically bind to PD-1 are disclosed in US 8,008,449 and WO2006/121168. Pidilizumab (CT-011; Cure Tech) is a humanized IgG1k monoclonal antibody that binds to PD-1. Peliezumab and other humanized anti-PD-1 monoclonal antibodies are disclosed in WO2009/101611. Pembrolizumab (formerly known as lambrolizumab, also known as MK03475; Merck) is a humanized IgG4 monoclonal antibody that binds to PD-1. Pacliizumab and other humanized anti-PD-1 antibodies are disclosed in US 8,354,509 and WO 2009/114335. MEDI 4736 (Medimmune) is a human monoclonal antibody that binds to PDL1 and inhibits ligand interaction with PD1. MDPL3280A (Genentech/Roche) is an IgG1 monoclonal antibody optimized for human Fc that binds to PD-L1. MDPL 3280A and other human monoclonal antibodies against PD-L1 are disclosed in U.S. Patent No. 7,943,743 and U.S. Patent Publication No. 20120039906. Other anti-PD-L1 binding agents include YW243.55.S70 (heavy and light chain variable regions are shown in SEQ ID NOs 20 and 21 of WO2010/077634) and MDX-1 105 (also known as BMS-936559, And an anti-PD-L1 binding agent such as disclosed in WO2007/005874). AMP-224 (B7-DCIg; Amplimmune; as disclosed in WO2010/027827 and WO2011/066342) is a PD-L2 Fc fusion soluble receptor that blocks the interaction between PD-1 and B7-H1. Other anti-PD-1 antibodies include AMP 514 (Amplimmune), particularly the anti-PD-1 antibodies disclosed in, for example, US 8,609,089, US 2010028330 and/or US 20120114649.

TIM3 (T-cell immunoglobulin-3) also negatively regulates T cell function, especially in CD4+ T helper 1 and CD8+ T cytotoxic 1 cells secreting IFN-g, and plays a key role in T cell depletion. Inhibition of the interaction between TIM3 and its ligands (eg, Galectin-9 (Gal9), phospholipid lysine (PS), and HMGB1) enhances the immune response. Antibodies, antibody fragments and other inhibitors of TIM3 and its ligands are available in the art and can be used in combination with the CD19 or BCMA CAR described herein. For example, an antibody, antibody fragment, small molecule or peptide inhibitor that targets TIM3 binds to the IgV domain of TIM3 to inhibit interaction with its ligand. Antibodies and peptides that inhibit TIM3 are disclosed in WO2013/006490 and US20100247521. Other anti-TIM3 antibodies include the humanized version of RMT3-23 (disclosed in Ngiow et al, 2011, Cancer Res, 71: 3540-3551) and the pure line 8B.2C12 (disclosed in Monney et al, 2002, Nature, 415: 536). -541)). Bispecific antibodies that inhibit TIM3 and PD-1 are disclosed in US20130156774.

In other embodiments, the agent that increases the cellular activity of the CAR is a CEACAM inhibitor (eg, CEACAM-1, CEACAM-3, and/or CEACAM-5 inhibitor). In one embodiment, the inhibitor of CEACAM is an anti-CEACAM antibody molecule. Exemplary anti-CEACAM-1 antibodies are described in WO 2010/125571, WO 2013/082366, WO 2014/059251, and WO 2014/022332, for example, monoclonal antibodies 34B1, 26H7, and 5F4; or recombinant forms thereof, such as, for example, US 2004 /0047858, US 7,132,255 and WO 99/052552. In other embodiments, the anti-CEACAM antibody binds to CEACAM-5, such as, for example, Zheng et al., PLoS One. September 2, 2010; 5(9).pii:e12529 (DOI: 10:1371/journal.pone .0021146), or cross-reacting with CEACAM-1 and CEACAM-5, as described, for example, in WO 2013/054331 and US 2014/0271618.

Without wishing to be bound by theory, it is believed that carcinoembryonic antigen cell adhesion molecules (CEACAM) such as CEACAM-1 and CEACAM-5 at least partially mediate inhibition of anti-tumor immune responses (see, eg, Markel et al, J Immunol. March 2002) . 15th; 168(6): 2803-10; Markel et al, J Immunol. November 1, 2006; 177(9): 6062-71; Markel et al., Immunology. February 2009; 126(2) :186-200; Markel et al, Cancer Immunol Immunother. February 2010; 59(2): 215-30; Ortenberg et al, Mol Cancer Ther. June 2012; 11(6): 1300-10; Stern et al, J Immunol, 2005 June 1; 174 (11):. 6692-701 ; Zheng et al., PLoS One 2010 of 9 Yue 2 Ri; 5 (9) .pii:. e12529). For example, CEACAM-1 has been described as a heterophilic ligand for TIM-3 and plays a role in TIM-3 mediated T cell tolerance and depletion (see, for example, WO 2014/022332; Huang et al. , (2014) Nature doi: 10.1038/nature13848). In the Examples, co-blocking of CEACAM-1 and TIM-3 has been shown to enhance anti-tumor immune responses in xenograft colorectal cancer models (see, for example, WO 2014/022332; Huang et al. (2014), supra). In other embodiments, co-blocking CEACAM-1 and PD-1 reduces T cell tolerance as described, for example, in WO 2014/059251. Thus, CEACAM inhibitors can be used with other immunomodulatory agents described herein (eg, anti-PD-1 and/or anti-TIM-3 inhibitors) to enhance against cancer, such as melanoma, lung cancer (eg, NSCLC), bladder The immune response of cancer, colon cancer, ovarian cancer, and other cancers as described herein.

LAG3 (lymphocyte activating gene-3 or CD223) is a cell surface molecule expressed on activated T cells and B cells, which has been shown to play a role in CD8+ T cell depletion. Antibodies, antibody fragments and other inhibitors of LAG3 and its ligands are available in the art and can be used in combination with the CD19 or BCMA CAR described herein. For example, BMS-986016 (Bristol-Myers Squib) is a monoclonal antibody that targets LAG3. IMP701 (Immutep) is an antagonist LAG3 antibody and IMP731 (Immutep and GlaxoSmithKline) is a depleted LAG3 antibody. Other LAG3 inhibitors include IMP321 (Immutep), a recombinant fusion protein of soluble fractions of LAG3 and Ig that bind to MHC class II molecules and activate antigen presenting cells (APCs). Other antibodies are disclosed, for example, in WO2010/019570.

In some embodiments, an agent that increases the cellular activity of a CAR can be, for example, comprises a fusion protein of a first domain and a second domain, wherein the first domain is a suppressor molecule or a fragment thereof, and the second domain is a polypeptide associated with a positive signal, eg, comprising an intracellular signaling construct as described herein Domain polypeptide. In some embodiments, a polypeptide associated with a positive signal can include a co-stimulatory domain of CD28, CD27, ICOS, such as an intracellular signaling domain of CD28, CD27, and/or ICOS, and/or a CD3, eg, as described herein. The main signaling domain. In one embodiment, the fusion protein is represented by the same cell that exhibits CAR. In another embodiment, the fusion protein is expressed by, for example, a cell that does not exhibit anti-BCMA CAR T cells or NK cells.

In one embodiment, the agent that enhances the cellular activity of the CAR described herein is miR-17-92.

In one embodiment, the agent that enhances the activity of the CAR described herein is a cytokine. Cytokines have important functions associated with T cell expansion, differentiation, survival, and homeostasis. Cytokines that can be administered to an individual receiving a cell exhibiting CAR as described herein include: IL-2, IL-4, IL-7, IL-9, IL-15, IL-18, and IL-21, or a combination thereof. In a preferred embodiment, the cytokine administered is IL-7, IL-15 or IL-21 or a combination thereof. The cytokine can be administered once a day or multiple times a day, for example twice a day, three times a day or four times a day. Cytokines can be administered for more than one day, for example, cytokine administration for 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 4 weeks. For example, cytokines are administered once a day for 7 days.

In an embodiment, the cytokine is administered in combination with a T cell expressing CAR. Cytokines can be administered simultaneously or in parallel with T cells expressing CAR, for example, on the same day. The cytokine can be prepared in the same pharmaceutical composition as the T cell expressing CAR, or can be prepared in a separate pharmaceutical composition. Alternatively, the cytokine can be administered shortly after administration of the T cell expressing the CAR, for example, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days or 7 days after administration of the T cell expressing the CAR. . In the embodiment in which the cytokine is administered in a dosing regimen that is administered over one day, the first day of the cytokine dosing regimen can be in the T with the performance of CAR The cells are administered on the same day, or the first day of the cytokine dosing regimen may be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days or 7 days after administration of the T cells expressing the CAR. In one embodiment, on day one, T cells expressing CAR are administered to the individual, and on the next day, the cytokines are administered once a day for 7 days. In a preferred embodiment, the cytokine to be administered in combination with a T cell expressing CAR is IL-7, IL-15 or IL-21.

In other embodiments, at least 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 4 weeks after administration of cells expressing CAR, for example, after administration of cells expressing CAR Cytokines are administered at months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 1 year or more. In one embodiment, the cytokine is administered after assessing the individual's response to cells expressing the CAR. For example, cells expressing CAR are administered to an individual according to the dosages and protocols described herein. Using any of the methods described herein, including inhibition of tumor growth, reduction of circulating tumor cells, or tumor regression, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks after administration of cells expressing CAR, Evaluation of individual cell therapy for CAR for 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 1 year or more reaction. Individuals that do not show sufficient response to cell therapy for CAR may be administered cytokines. Cytokine administration increases the cellular or anti-cancer activity of CAR in individuals who have a suboptimal response to cell therapy that expresses CAR. In a preferred embodiment, the cytokine administered after administration of cells expressing CAR is IL-7.

Combined with a low immune booster dose of mTOR inhibitor

The methods described herein use a low immunopotentiating dose of an mTOR inhibitor, such as an additional mTOR inhibitor, including a rapamycin analogue, such as RAD001. Administration of a low immunopotentiating dose of an mTOR inhibitor (e.g., a dose sufficient to completely inhibit the immune system but sufficient to enhance immune function) may optimize the efficacy of immune effector cells, such as T cells or cells expressing CAR, in an individual. Methods, dosages, treatment regimens, and suitable pharmaceutical compositions for measuring mTOR inhibition are described in U.S. Patent Application Serial No. 2015/01240036.

In one embodiment, administering a low immunopotentiating dose of the mTOR inhibitor results in one or more of: i) a decrease in the number of PD-1 positive immune effector cells; ii) an increase in the number of PD-1 negative immune effector cells ; iii) an increase in the ratio of PD-1 negative immune effector cells/PD-1 positive immune effector cells; iv) an increase in the number of primary T cells; v) for example in the following markers on memory T cells such as memory T cell precursor cells; Increased performance of one or more: CD62L ^high , CD127 ^high , CD27 ⁺ and BCL2; vi) decreased expression of KLRG1 on, for example, memory T cells such as memory T cell precursor cells; or vii) increased number of memory T cell precursor cells , the cells having a cell, for example, any of the following features or a combination of: CD62L increased ^high, CD127 ^high increase, CD27 ⁺ increase, decrease and increase of BCL2 KLRG1; and wherein, for example, as compared to the untreated individual, or more Any of these, such as i), ii), iii), iv), v), vi) or vii), for example, occurs at least briefly.

In another embodiment, administration of a low immunopotentiating dose of an mTOR inhibitor elicits a cell that exhibits CAR, such as culture or proliferation in an individual, for example, as compared to an untreated CAR-expressing cell or an untreated individual. Increase or increase in persistence. In the examples, the increase in proliferation or persistence is associated with an increase in the number of cells expressing CAR. Methods for measuring the increase or extension of proliferation are described in Examples 15 and 16. In another embodiment, administration of a low immunopotentiating dose of an mTOR inhibitor elicits, for example, cells expressing CAR in culture or in an individual versus cancer cells, as compared to untreated cells expressing CAR or untreated individuals, for example The killing increases. In an embodiment, increased cancer cell killing is associated with a decrease in tumor volume. Herein, for example, in Examples 2, 5-6, 8 and 13, for measuring an increase in cancer cell killing method. In one embodiment, a cell expressing a CAR molecule, such as a CAR molecule described herein, is administered in combination with a low immunopotentiating dose of an mTOR inhibitor, such as an additional mTOR inhibitor, such as RAD001 or a catalytic mTOR inhibitor. For example, administration of a low immunopotentiating dose of an mTOR inhibitor can be initiated prior to administration of a cell that exhibits CAR as described herein; ending prior to administration of a cell expressing CAR as described herein; The cells expressing the CAR start simultaneously; overlap with the cells expressing the CAR as described herein; or continue after administration of the cells expressing CAR as described herein.

Alternatively or additionally, administration of a low immunopotentiating dose of mTOR inhibitor optimizes immune effector cells to be engineered to exhibit the CAR molecules described herein. In such embodiments, a low immunopotentiating dose of an mTOR inhibitor, such as an allosteric inhibitor, such as RAD001 or a catalytic inhibitor, is harvested from an individual to be engineered to express the immune effector cells of the CAR molecule described herein. For example, T cells or NK cells start or end before.

In another embodiment, for example, an immune effector cell, such as a T cell or an NK cell, to be engineered to express a CAR molecule as described herein after harvesting from an individual, or an immune effector cell that exhibits CAR, eg, prior to administration to an individual, For example, T cells or NK cells can be cultured in the presence of a low immunopotentiating dose of mTOR inhibitor.

In one embodiment, administering to the individual a low immunopotentiating dose of the mTOR inhibitor comprises, for example, once a week, eg, in an immediate release dosage form, administered at 0.1 to 20, 0.5 to 10, 2.5 to 7.5, 3 to 6, or about 5 mg. RAD001 or its bioequivalent dose. In one embodiment, administering to the individual a low immunopotentiating dose of the mTOR inhibitor comprises, for example, once a week, for example in a sustained release dosage form, administered at 0.3 to 60, 1.5 to 30, 7.5 to 22.5, 9 to 18 or about 15 mg. RAD001 or its bioequivalent dose.

In one embodiment, the dose of the mTOR inhibitor causes or provides at least 5% but no more than 90%, at least 10% but no more than 90%, at least 15% but no more than 90%, at least 20% but no more than 90%, At least 30% but not more than 90%, at least 40% but not more than 90%, at least 50% but not more than 90%, at least 60% but not more than 90%, at least 70% but not more than 90%, at least 5% but not more than 80%, at least 10% but not more than 80%, at least 15% but not more than 80%, at least 20% but not more than 80%, at least 30% but not more than 80%, at least 40% but not more than 80%, at least 50% but not more than 80%, at least 60% but not more than 80%, at least 5% but not more than 70%, at least 10% but not more than 70%, at least 15% but not more than 70%, at least 20 % but not more than 70%, at least 30% but not more than 70%, at least 40% but not more than 70%, at least 50% but not more than 70%, at least 5% but not more than 60%, at least 10% but not more than 60 %, at least 15% but not more than 60%, at least 20% but not more than 60%, at least 30% but not more than 60%, at least 40% but not more than 60%, at least 5% but not more than 50%, at least 10% But not more than 50%, at least 15% but not more than 50%, at least 20% but not more than 50%, at least 30% but not more than 50%, at least 40% but not more than 50%, at least 5% but not more than 40% , at least 10% but not more than 40%, at least 15% but not more than 40%, at least 20% but not more than 40%, at least 30% but not more than 40%, or at least 35% but not more than 40%, at least 5% But not more than 30%, at least 10% but not more than 30%, at least 15% but not more than 30%, at least 20% but not more than 30%, or at least 2 5% but not more than 30% of mTOR inhibition.

In one embodiment, administering to the individual a low immunopotentiating dose of the mTOR inhibitor comprises, for example, once a week, eg, in an immediate release dosage form, administered at 0.1 to 20, 0.5 to 10, 2.5 to 7.5, 3 to 6, or about 5 mg. RAD001 or its bioequivalent dose. In one embodiment, administering to the individual a low immunopotentiating dose of the mTOR inhibitor comprises, for example, once a week, for example in a sustained release dosage form, administered at 0.3 to 60, 1.5 to 30, 7.5 to 22.5, 9 to 18 or about 15 mg. RAD001 or its bioequivalent dose.

The degree of inhibition of mTOR can be communicated to or correspond to the degree of inhibition of P70 S6 kinase, for example, the degree of mTOR inhibition can be determined by the degree of P70 S6 kinase activity reduction, for example, by a decrease in phosphorylation of P70 S6 kinase. The degree of mTOR inhibition can be assessed by a variety of methods, such as described in U.S. Patent Application Serial No. U.S. Patent No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. The P70 S6 kinase activity was measured by Boulay analysis; the phosphoric acid was measured by Western blotting method. The degree of S6; or the change in the ratio of PD1 negative immune effector cells to PD1 positive immune effector cells.

As used herein, the term "mTOR inhibitor" refers to a compound or ligand that inhibits mTOR kinase in a cell, or a pharmaceutically acceptable salt thereof. In one embodiment, the mTOR inhibitor is an ectopic inhibitor. Ectopic mTOR inhibitors include the neutral tricyclic compound rapamycin (sirolimus), a compound associated with rapamycin, that is, a compound having a structure and function similar to rapamycin, including, for example, Rapamycin derivatives, rapamycin analogues (also known as Reparog) and other macrolide compounds that inhibit mTOR activity. In one embodiment, the mTOR inhibitor is a catalytic inhibitor.

Rapamycin is a known macrolide antibiotic produced by Streptomyces hygroscopicus having the structure shown in Formula A.

See, for example, McAlpine, J. B. et al., J. Antibiotics (1991) 44: 688; Schreiber, S. L. et al., J. Am. Chem. Soc. (1991) 113:7433; U.S. Patent No. 3,929,992. There are various numbering schemes proposed for rapamycin. To avoid confusion, when a particular rapamycin analogue is named herein, the names are given with reference to rapamycin using the numbering scheme of Formula A.

A rapamycin analogue suitable for use in the present invention is, for example, an O-substituted analog wherein the hydroxy group on the cyclohexyl ring of rapamycin is substituted with OR ₁ wherein R ₁ is a hydroxyalkyl group, a hydroxyalkoxy alkane A, decylaminoalkyl or aminoalkyl; for example, RAD001, also known as everolimus, as described in US 5,665,772 and WO 94/09010, each of which is incorporated herein by reference.

Other suitable rapamycin analogues include analogs substituted at position 26 or 28. The rapamycin analogue may be the epimer of the above analog, especially the epimer of the substituted analog at position 40, 28 or 26, and may optionally be further hydrogenated, for example as in US 6,015,815, WO 95/14023 and WO 99/15530, the contents of which are incorporated by reference; for example, ABT 578, also known as zotarolimus or rapamycin, as described in US 7,091,213, WO 98/02441 and WO 01/14387 The content of which is incorporated by reference; for example, AP23573, also known as diphosphomethoxazole.

Examples of rapamycin analogues from US 5,665,772 suitable for use in the present invention include, but are not limited to, 40-O-benzyl-rapamycin, 40-O-(4'-hydroxymethyl)benzamide -Rapamycin, 40-O-[4'-(1,2-dihydroxyethyl)]benzyl-rapamycin, 40-O-allyl-rapamycin, 40- O-[3'-(2,2-dimethyl-1,3-dioxolan-4(S)-yl)-propan-2'-ene-1'-yl]-rapamycin, (2'E,4'S)-40-O-(4',5'-dihydroxypent-2'-ene-1'-yl)-rapamycin, 40-O-(2-hydroxy)ethoxylate Carbonylmethyl-rapamycin, 40-O-(2-hydroxy)ethyl-rapamycin, 40-O-(3-hydroxy)propyl-rapamycin, 40-O-(6- Hydroxy)hexyl-rapamycin, 40-O-[2-(2-hydroxy)ethoxy]ethyl-rapamycin, 40-O-[(3S)-2,2-dimethyldi Oxypentan-3-yl]methyl-rapamycin, 40-O-[(2S)-2,3-dihydroxypropan-1-yl]-rapamycin, 40-O-(2- Ethyloxy)ethyl-rapamycin, 40-O-(2-nicotinyloxy)ethyl-rapamycin, 40-O-[2-(N-morpholinyl)醯oxy]ethyl-rapamycin, 40-O-(2-N-imidazolylethoxy)ethyl-rapamycin, 40-O-[2-(N-methyl-N '-Piperazinyl)ethyloxy]ethyl-rapamycin, 39 -O-desmethyl-39,40-O, O-extended ethyl-rapamycin, (26R)-26-dihydro-40-O-(2-hydroxy)ethyl-rapamycin, 40-O-(2-Aminoethyl)-rapamycin, 40- O-(2-acetamidoethyl)-rapamycin, 40-O-(2-nicotinoguanidinoethyl)-rapamycin, 40-O-(2-(N-A) Base-imidazole-2'-ylethoxycarbonylamino)ethyl)-rapamycin, 40-O-(2-ethoxycarbonylaminoethyl)-rapamycin, 40-O-(2 -tolylsulfonylaminoethyl)-rapamycin and 40-O-[2-(4',5'-diethoxycarbonyl-1',2',3'-triazole-1'-- Base)-ethyl]-rapamycin.

Other rapamycin analogues which are known to be suitable for use in the present invention are hydroxy groups on the cyclohexyl ring of rapamycin and/or analogs in which the hydroxy group at position 28 is replaced by a hydroxyester group, as described, for example, in US RE 44,768. A rapamycin analogue, such as temsirolimus.

Other rapamycin analogues suitable for use in the present invention include such analogs wherein the methoxy group at position 16 is preferably (optionally hydroxyl substituted) alkynyloxy, benzyl, o-methoxy Substitution of another substituent of a benzyl or chlorobenzyl group, and/or deletion of a methoxy group at position 39 together with 39 carbon causes the cyclohexyl ring of rapamycin to become a position 39 methyloxy group Cyclopentyl rings; such analogs are as described, for example, in WO 95/16691 and WO 96/41807, the contents of which are hereby incorporated by reference. The analog can be further modified to alkylate the hydroxy group at position 40 of rapamycin and/or reduce the 32-carbonyl group.

Rapamycin analogs from WO 95/16691 include, but are not limited to, 16-desmethoxy-16-(pent-2-ynyl)oxy-rapamycin, 16-desmethoxy-16 -(but-2-ynyl)oxy-rapamycin, 16-desmethoxy-16-(propargyl)oxy-rapamycin, 16-desmethoxy-16-(4 -hydroxy-but-2-ynyloxy-rapamycin, 16-desmethoxy-16-benzyloxy-40-O-(2-hydroxyethyl)-rapamycin, 16 -desmethoxy-16-benzyloxy-rapamycin, 16-desmethoxy-16-o-methoxybenzyl-rapamycin, 16-desmethoxy-40-O -(2-methoxyethyl)-16-pent-2-ynyl)oxy-rapamycin, 39-desmethoxy-40-deoxy-39-methylindenyl-42-decane Rapamycin, 39-desmethoxy-40-deoxy-39-hydroxymethyl-42-norderapmycin, 39-desmethoxy-40-deoxy-39-carboxy-42 - rapamycin, 39-desmethoxy-40-deoxy-39-(4-methyl-piperazin-1-yl)carbonyl-42-nortropin, 39-demethoxy -40-desoxy-39-(morpholin-4-yl)carbonyl-42-northrazole, 39-desmethoxy-40-deoxy --39-[N-methyl, N-(2-pyridin-2-yl-ethyl)]amine-carbamoyl-42-norderapmycin and 39-desmethoxy-40-deoxy -39-(p-toluenesulfonylfluorenylmethyl)-42-norderapmycin.

Rapamycin analogs from WO 96/41807 include, but are not limited to, 32-deoxy-rapamycin, 16-O-pent-2-ynyl-32-deoxy-rapamycin, 16- O-pent-2-ynyl-32-deoxy-40-O-(2-hydroxy-ethyl)-rapamycin, 16-O-pent-2-ynyl-32-(S)-di Hydrogen-40-O-(2-hydroxyethyl)-rapamycin, 32(S)-dihydro-40-O-(2-methoxy)ethyl-rapamycin and 32(S) - Dihydro-40-O-(2-hydroxyethyl)-rapamycin.

Another suitable rapamycin analogue is umirolimus as described in US 2005/0101624, the contents of which are incorporated by reference.

RAD001 is also known as Afinitor®, which has the chemical name (1R, 9S, 12S, 15R, 16E, 18R, 19R, 21R, 23S, 24E, 26E, 28E, 30S, 32S, 35R)-1 ,18-Dihydroxy-12-{(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl }-19,30-Dimethoxy-15,17,21,23,29,35-hexamethyl-11,36-dioxa-4-aza-tricyclo[30.3.1.04,9]6 (triacontane)-16,24,26,28-tetraene-2,3,10,14,20-pentanone, as in US 5,665,772 and WO 94/09010 (incorporated by reference) Said.

Other examples of ectopic mTOR inhibitors include sirolimus (rapamycin, AY-22989), 40-[3-hydroxy-2-(hydroxymethyl)-2-methylpropionate]-rapa Taxomycin (also known as temsirolimus or CCI-779) and dexamethasone (AP-23573/MK-8669). Other examples of ectopic mTor inhibitors include zotarolimus (ABT 578) and umamimus.

Alternatively or additionally, it has been discovered that a catalytic ATP-competitive mTOR inhibitor directly targets the mTOR kinase domain and targets both mTORC1 and mTORC2. These are also mTORC1 inhibitors that are more potent than ectopic mTOR inhibitors such as rapamycin because they modulate anti-rapamycin mTORC1 output, such as 4EBP1-T37/46 phosphorylation and cap-dependent translation.

Catalytic inhibitors include: BEZ235 or 2-methyl-2-[4-(3-methyl-2-oxo-8-quinoline Benz-3-yl-2,3-dihydro-imidazo[4,5-c]quinolin-1-yl)-phenyl]-propionitrile, or monotosylate form (synthesis of BEZ235 is described in WO2006/122806); CCG168 (also known as AZD-8055, Chresta, CM et al, Cancer Res, 2010, 70(1), 288-298), which has the chemical name {5-[2,4-double- ((S)-3-methyl-morpholin-4-yl)-pyrido[2,3d]pyrimidin-7-yl]-2-methoxy-phenyl}-methanol; 3-[2,4 - bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl]-N-methylbenzimidamide (WO09104019); 3-(2 -aminobenzo[d]oxazol-5-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (WO 10051043 and WO2013023184); N-(3- (N-(3-((3,5-Dimethoxyphenyl)amino)quinoxalin-2-yl)amine sulfonyl)phenyl)-3-methoxy-4-methylbenzene Formamide (WO07044729 and WO12006552); PKI-587 (Venkatesan, AM, J. Med. Chem., 2010, 53, 2636-2645), which has the chemical name 1-[4-[4-(dimethylamine) Piperidin-1-carbonyl]phenyl]-3-[4-(4,6-dimorpholine)-1,3,5-triazin-2-yl)phenyl]urea; GSK-2126458 ( ACS Med. Chem. Lett., 2010, 1, 39-43) having the chemical name 2,4-difluoro-N-{2-methoxy-5-[4-(4-pyridazinyl)- 6-quinolinyl]-3-pyridyl Pyridyl}benzenesulfonamide; 5-(9-isopropyl-8-methyl-2-morpholinyl-9H-indol-6-yl)pyrimidin-2-amine (WO10114484); and (E)- N-(8-(6-Amino-5-(trifluoromethyl)pyridin-3-yl)-1-(6-(2-cyanopropan-2-yl)pyridin-3-yl)-3 -Methyl-1H-imidazo[4,5-c]quinolin-2(3H)-ylidene cyanamide (WO12007926).

Further examples of catalytic mTOR inhibitors include 8-(6-methoxy-pyridin-3-yl)-3-methyl-1-(4-piperazin-1-yl-3-trifluoromethyl-phenyl )-1,3-Dihydro-imidazo[4,5-c]quinolin-2-one (WO2006/122806) and Ku-0063794 (Garcia-Martinez JM et al, Biochem J., 2009, 421 (1) ), 29-42). Ku-0063794 is a mammalian target-specific inhibitor of rapamycin (mTOR). WYE-354 is another example of a catalytic mTOR inhibitor (Yu K et al, (2009). Biochemical, Cellular, and In vivo Activity of Novel ATP-Competitive and Selective Inhibitors of the Mammalian Target of Rapamycin. Cancer Res. 69 ( 15): 6232-6240).

MTOR inhibitors suitable for use in accordance with the invention also include prodrugs, derivatives, and pharmaceuticals thereof. An acceptable salt or an analog of any of the foregoing.

An mTOR inhibitor such as RAD001 can be formulated to facilitate delivery based on the particular dosages described herein based on methods recognized in the art. In particular, U.S. Patent No. 6,004,973, the disclosure of which is incorporated herein by reference in its entirety in its entirety in its entirety in its entirety in the in the in the in the in the

Methods and biomarkers for assessing CAR effectiveness or sample suitability

In another aspect, the invention provides an assessment or monitoring of the effectiveness or sample (eg, a clearance sample) of a cell therapy (eg, a BCMACAR therapy) that exhibits CAR in an individual (eg, an individual having a cancer, such as a blood cancer) A method of suitability for CAR therapy (eg, BCMACAR therapy). The method includes obtaining a value for CAR therapy effectiveness or sample suitability, wherein the value indicates the effectiveness or suitability of a cell therapy that exhibits CAR.

In an embodiment, the value of the effectiveness or sample suitability of the CAR therapy comprises measurements of one, two, three, four, five, six or more (all): (i) sample Resting T _EFF cells, resting T _REG cells, younger T cells (eg, younger CD4 or CD8 cells or γ/δ T cells) or early memory T cells (eg, a sample of a cleanup or a sample of a cell product produced to produce CAR) The content or activity of one, two, three or more (eg, all) or a combination thereof; (ii) activated T _EFF cells in a sample (eg, a scavenging sample or a cell product sample that produces CAR) , activating _TECG cells, the content or activity of one, two, three or more (eg, all) of the older T cells (eg, older CD4 or CD8 cells) or advanced memory T cells, or a combination thereof; (iii) an immune cell depletion marker, such as one, two or more checkpoint inhibitors (eg, PD-1, PD-L1, in a sample (eg, a cleanup sample or a sample of a cell product that produces a CAR). The content or activity of TIM-3 and/or LAG-3). In one embodiment, the immune cells have a depleted phenotype, eg, a total of at least two depletion markers, eg, a total of PD-1 and TIM-3. In other embodiments, the immune cells have a depleted phenotype, eg, a total of at least two depletion markers, eg, a total of PD-1 and LAG-3; (iv) a sample (eg, a scavenging sample or manufacturing performance CAR) The content or activity of CD27 and/or CD45RO- (eg, CD27+CD45RO-) immune effector cells in a CD4+ or CD8+ T cell population; (v) selected from CCL20, IL-17a and/or IL-6. , one of the biomarkers of PD-1, PD-L1, LAG-3, TIM-3, CD57, CD27, CD122, CD62L, and KLRG1, two, three, four, five, ten, two Content or activity of ten or more; (vi) a sample of a cell product exhibiting CAR, such as a cytokine content or activity in a sample of a cell product exhibiting BCMA CAR (eg, a quality of a cytokine library); or (vii) manufactured Transduction efficiency of cells expressing CAR in a cell product sample of CAR.

In some embodiments of any of the methods disclosed herein, the CAR-presenting cell therapy comprises a plurality (eg, a population) of immune effector cells that exhibit CAR, eg, a plurality (eg, a population of) T cells or NK cells, or a combination thereof. In one embodiment, the cell therapy that represents CAR is BCMACAR therapy.

In some embodiments of any of the methods disclosed herein, the measurement of one or more of (i)-(vii) is obtained from a sample obtained from an individual. The purge sample can be evaluated prior to infusion or reinfusion.

In some embodiments of any of the methods disclosed herein, the measurement of one or more of (i)-(vii) is obtained from a sample of a cell product that is manufactured to represent CAR, such as a sample of a cell product that exhibits BCMACAR. The cellular product of the manufactured CAR can be evaluated prior to infusion or reinfusion.

In some embodiments of any of the methods disclosed herein, the individual is evaluated during or after acceptance of the cell therapy that exhibits CAR.

In some embodiments of any of the methods disclosed herein, the measuring of one or more of (i)-(vii) assesses one or more of gene expression, flow cytometry, or protein expression Type of person.

In some embodiments of any of the methods disclosed herein, the method further comprises identifying the individual as a responder, a non-responder, a relapser or a non-based based on the measurement of one or more of (i)-(vii) Recurrence.

In some embodiments of any of the methods disclosed herein, the responder (eg, a complete responder) has or is identified to have one, two or more of GZMK, PPF1BP2, or naive T cells as compared to a non-reactive person (all) greater content or activity.

In some embodiments of any of the methods disclosed herein, the non-reactive person has or is identified to have IL22, IL-2RA, IL-21, IRF8, IL8, CCL17, CCL22, effector T cells or Regulates the greater content or activity of one, two, three, four, five, six, seven or more (all) of T cells.

In one embodiment, the relapser is an amount of one or more (eg, 2, 3, 4, or all) of the following genes having or identified as having an increase compared to the non-recurrent: MIR199A1, MIR1203, Uc021ovp, ITM2C, and HLA-DQB1, and/or one or more of the following genes reduced compared to non-relapsers (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11) Or all of the patients in the performance: PPIAL4D, TTTY10, TXLNG2P, MIR4650-1, KDM5D, USP9Y, PRKY, RPS4Y2, RPS4Y1, NCRNA00185, SULT1E1 and EIF1AY.

In some embodiments of any of the methods disclosed herein, the complete responder has or is identified to have a greater percentage, such as a statistically significant greater percentage, than a reference value, such as a percentage of non-responders of CD8+ T cells. CD8+ T cells.

In some embodiments of any of the methods disclosed herein, the complete responder has or is identified to have a greater percentage of CD27+ in the CD8+ population than the number of non-responders with reference values, such as CD27+CD45RO-immunore effector cells. CD45RO - immune effector cells.

In some embodiments of any of the methods disclosed herein, the complete responder or portion The responder has or is identified to have a greater, e.g., statistically significant, percentage of CD4+ T cells compared to a reference value, such as the percentage of non-responders of CD4+ T cells.

In some embodiments of any of the methods disclosed herein, the complete responder has or is identified to have a reference value, such as a resting T _EFF cell, a resting T _REG cell, a younger T cell (eg, a younger CD4 or CD8 cell). Or a greater percentage of non-responders of early memory T cells, a greater percentage of resting T _EFF cells, resting T _REG cells, younger T cells (eg, younger CD4 or CD8 cells or γ/δ T cells) or early memory T One, two, three or more (eg, all) of the cells, or a combination thereof.

Herein disclosed a number of methods of of any one of embodiments embodiment, non-responders have or the identified with a reference value, such as activated T _{the EFF} cells, activated T _REG cells, older T cells (e.g. older CD4 or CD8 cells) Or a greater percentage of responders in late memory T cells, a greater percentage of activated T _EFF cells, activated T _REG cells, older T cells (eg, older CD4 or CD8 cells) or one of late memory T cells, two , three or more (eg all) or a combination thereof.

In some embodiments of any of the methods disclosed herein, the non-reactive person has or is identified with a greater percentage of immune cell depletion markers, such as one, two or more immunological checkpoint inhibitors (eg, PD) -1, PD-L1, TIM-3 and/or LAG-3). In one embodiment, the non-responder has or is identified to have a greater percentage of PD-1, PD-L1 or LAG compared to the percentage of immune effector cells from the responder that exhibit PD-1 or LAG-3. -3 immune effector cells (eg, CD4+ T cells and/or CD8+ T cells) (eg, CD4+ cells and/or CD8+ T cells expressing CAR).

In one embodiment, the non-responder has or is identified with a greater percentage of immune cells having a depleted phenotype, eg, a total of at least two depletion markers, eg, a total of PD-1, PD-L1, and/or Or immune cells of TIM-3. In other embodiments, the non-responder has or is identified with a greater percentage of immune cells having a depleted phenotype, eg, a total of at least two depletion markers, eg, a total of PD-1 and LAG-3 immunoassay cell.

In some embodiments of any of the methods disclosed herein, the non-reactive person has or is identified as having a population of cells expressing the CAR (eg, a BCMACAR+ cell population) compared to a responder (eg, a complete responder) of a cell therapy that exhibits CAR A larger percentage of PD-1/PD-L1+/LAG-3+ cells.

In some embodiments of any of the methods disclosed herein, a portion of the responders have or have been identified to have a higher percentage of PD-1/PD- in a population of cells exhibiting CAR (eg, BCMACAR+ cell population) compared to the responder. L1+/LAG-3+ cells.

In some embodiments of any of the methods disclosed herein, the non-reactive person has or is identified to have a depleted phenotype of PD1/PD-L1+CAR+ and a LAG3 in a cell population exhibiting CAR (eg, BCMACAR+ cell population) A total performance.

In some embodiments of any of the methods disclosed herein, the non-responder has or is identified to have a greater percentage of the population of cells expressing the CAR (eg, BCMA CAR+ cell population) than the responder (eg, a complete responder) PD-1/PD-L1+/TIM-3+ cells.

In some embodiments of any of the methods disclosed herein, a portion of the responders have or have been identified to have a higher percentage of PD-1/PD- in a population of cells exhibiting CAR (eg, BCMACAR+ cell population) compared to the responder. L1+/TIM-3+ cells.

In some embodiments of any of the methods disclosed herein, the presence of CD8+CD27+CD45RO- T cells in the depletion sample is a predictor of the individual's positive response to a cell therapy (eg, BCMACAR therapy) that exhibits CAR.

In some embodiments of any of the methods disclosed herein, a high percentage of PD1+CAR+ and LAG3+ or TIM3+ T cells in a depletion sample is a predictor of an individual's poor response to a cell therapy (eg, BCMACAR therapy) that exhibits CAR.

In some embodiments of any of the methods disclosed herein, the responder (eg, a full or partial responder) has one, two, three or more (or all) of the following types: (i) A greater number of CD27+ immune effector cells than a reference value, such as the number of non-responders of CD27+ immune effector cells; (ii) a greater number than a reference value, such as the number of non-responders of CD8+ T cells CD8+ T cells; (iii) having a lower number of one or more checkpoint inhibitors, such as selected from PD-, compared to a reference value, such as the number of non-responders of cells exhibiting one or more checkpoint inhibitors 1. Immunological cells of PD-L1, LAG-3, TIM-3 or KLRG-1 or a combination of checkpoint inhibitors; or (iv) with reference values, such as resting T _EFF cells, resting T _REG cells, primitive CD4 cells Larger numbers of static T _EFF cells, resting T _REG cells, primitive CD4 cells, unstimulated memory T cells, or early memory compared to the number of unstimulated memory T cells or early memory T cells. One of the T cells, two, three, four or four Above (all) or a combination thereof.

In some embodiments of any of the methods disclosed herein, the cytokine content or activity of (vi) is selected from the group consisting of cytokines CCL20/MIP3a, IL17A, IL6, GM-CSF, IFNy, IL10, IL13, IL2, IL21, One, two, three, four, five, six, seven, eight or more (or all) of IL4, IL5, IL9 or TNFα or a combination thereof. The cytokine may be selected from one of IL-17a, CCL20, IL2, IL6 or TNFa, two, three, four or more (all). In one embodiment, an increase in the amount or activity of a cytokine selected from one or both of IL-17a and CCL20 is indicative of an increase in response or a decrease in relapse.

In some embodiments of any of the methods disclosed herein, a transduction efficiency of 15% or greater in (vii) indicates an increase in response or a decrease in relapse.

In some embodiments of any of the methods disclosed herein, less than 15% of the transduction efficiency in (vii) indicates a decrease in response or an increase in relapse.

In embodiments, responders, non-responders, relapsers, or non-relapsers identified by the methods herein can be further evaluated according to clinical criteria. For example, a complete responder has or is identified as having a disease, such as cancer, that shows complete response to treatment, such as an individual with complete remission. For example, using such as NCCN Guidelines ^® or Cheson person, J Clin Oncol 17: 1244 ( 1999) and Cheson et al., "Revised Response Criteria for Malignant Lymphoma", J Clin Oncol 25: 579-586 ( 2007) ( incorporated by reference are The way to do this is to identify complete responses. Some responders have or are identified as having a disease, such as cancer, a partial response to treatment, such as a partial remission. Partial reactions can be identified, for example, using the NCCN Guidelines® or Cheson criteria as described herein. A non-responder suffers or is identified as having a disease, such as cancer, an individual who does not show a response to treatment, such as a patient having a stable disease or a progressive disease. Non-responders can be identified, for example, using the NCCN Guidelines® or Cheson criteria as described herein.

Alternatively, or in combination with the methods disclosed herein, in response to the value, one, two, three, four or more of the following are performed: for example, a cell expressing CAR is administered to a responder or a non-relapser Therapy; administration of a cell therapy that alters the performance of a dose of CAR; changes in the progress or time course of a cell therapy that exhibits CAR; for example, administration of another agent to a non-responder or part of a responder to a cell therapy that exhibits CAR, such as checkpoint suppression Agents, such as combinations of checkpoint inhibitors described herein; administering to a non-responder or a portion of a responder a treatment that increases the number of younger T cells in an individual prior to treatment with a cell therapy that exhibits CAR; modifying cells expressing CAR Methods of manufacturing a therapy, such as enriching younger T cells prior to introduction of a nucleic acid encoding a CAR, or, for example, for individuals identified as non-responders or partial responders, increasing transduction efficiency; for example, for non-responders or partial responders or Recurrence, administration of alternative therapy; or if the individual is or is identified as non-responder or relapse, for example, by depletion of CD25, administration of cyclophosphamide, anti-GITR antibody or One or more of the co-reducing T _REG cell population and / or genetic characteristics T _REG.

In certain embodiments, the individual is pre-treated with an anti-GITR antibody. In one embodiment, the individual is treated with an anti-GITR antibody prior to infusion or reinfusion.

Biopolymer delivery method

In some embodiments, one or more cells exhibiting CAR as disclosed herein can be administered or delivered to an individual via a biopolymer scaffold, such as a biopolymer implant. The biopolymer scaffold can support or enhance the delivery, amplification, and/or dispersion of the CAR-expressing cells described herein. Biopolymer scaffolds comprise polymers that are naturally or synthetically biocompatible (eg, do not substantially induce inflammatory or immune responses) and/or biodegradable.

Examples of suitable biopolymers include, but are not limited to, agar, agarose, alginate, alginate/calcium phosphate cement (CPC), beta, alone or in combination with any other polymer composition at any concentration and in any ratio. -galactosidase (β-GAL), (1,2,3,4,6-pentaethyl-a-galactose), cellulose, chitin, polyglucamine, collagen, elastin, Gelatin, hyaluronic acid, collagen, hydroxyapatite, poly(3-hydroxybutyrate-co-3-hydroxy-hexanoate) (PHBHHx), poly(lactide), poly(caprolactone) (PCL ), poly(lactide-co-glycolide) (PLG), polyethylene oxide (PEO), poly(lactic-co-glycolic acid) (PLGA), polyoxypropylene (PPO), polyvinyl alcohol (PVA) ), silk, soy protein and isolated soy protein. The biopolymer may be a molecule that promotes adhesion or migration, such as a collagen mimetic peptide that binds to a collagen receptor of lymphocytes and/or enhances the transmission, amplification or function of a cell to be delivered, such as a stimulating molecule for anticancer activity. Strengthen or upgrade. The biopolymer scaffold can be an injectable, such as a gel or semi-solid, or a solid composition.

In some embodiments, the CAR-expressing cells described herein are seeded onto a biopolymer scaffold prior to delivery to an individual. In embodiments, the biopolymer scaffold further comprises one or more other therapeutic agents described herein (eg, another cell, antibody, or small molecule that exhibits CAR) or enhanced performance, such as a biopolymer incorporated or bound to the scaffold. An agent that activates the cells of CAR. In an embodiment, the biopolymer scaffold is, for example, injected intratumorally or surgically into a tumor or implanted in a location near the tumor sufficient to mediate anti-tumor effects. Other examples of biopolymer compositions and methods for their delivery are described in Stephan et al, Nature Biotechnology , 2015, 33: 97-101; and WO 2014/110591.

Pharmaceutical composition and treatment

The pharmaceutical compositions of the present invention may comprise cells expressing CAR (e.g., a plurality of cells expressing CAR as described herein) and one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. combination. Such compositions may contain buffering agents such as neutral buffered saline, phosphate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides Or an amino acid such as glycine; an antioxidant; a chelating agent such as EDTA or glutathione; an adjuvant such as aluminum hydroxide; and a preservative. In one aspect, the compositions of the invention are formulated for intravenous administration.

The pharmaceutical composition of the present invention can be administered in a manner suitable for the treatment (or prevention) of the disease. Although the appropriate dose can be determined by clinical trials, the amount and frequency of administration will be determined by factors such as the condition of the patient and the type and severity of the patient's condition.

In one embodiment, the pharmaceutical composition is substantially free, such as in the absence of detectable levels of contaminants, for example selected from the group consisting of endotoxin, mycoplasma, replication competent lentivirus (RCL) , p24, VSV-G nucleic acid, HIV gag, residual anti-CD3/anti-CD28 coated beads, mouse antibody, pooled human serum, bovine serum albumin, bovine serum, medium components, vector-encapsulated cells or Body composition, bacteria and fungi. In one embodiment, the bacterium is at least one selected from the group consisting of Alcaligenes faecalis, Candida albicans, Escherichia coli, Haemophilus influenza. , Neisseria meningitides, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumonia, and Streptococcus pyogenes group A.

When the "immune effective amount", the "antitumor effective amount", the "tumor inhibition effective amount" or the "therapeutic amount" is indicated, the precise amount of the composition of the present invention to be administered can be considered by the physician in consideration of the age, body weight, and tumor of the individual. The size, the degree of infection or cancer metastasis, and the difference in patient (individual) conditions are determined. It is generally possible to specify that a pharmaceutical composition comprising a T cell as described herein can be from 10 ⁴ to 10 ⁹ cells per kilogram of body weight, in some cases from 10 ⁵ to 10 ⁶ cells per kilogram of body weight (including all integer values in their range) The dose is administered. The T cell composition can also be administered in multiple doses at this dose. Cells can be administered by using infusion techniques commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988).

In certain aspects, it may be desirable to administer activated T cells to an individual, followed by re-blood (or clearance), from which T cells are activated, and with such activated and expanded T cells. Infusion of the patient. This process can be performed multiple times every few weeks. In some aspects, T cells can be activated from 10 cc to 400 cc of blood drawn. In some aspects, T cells are activated from 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100 cc of blood drawn.

The subject composition can be administered in any suitable manner, including by aerosol inhalation, injection, ingestion, infusion, implantation or transplantation. The compositions described herein can be administered to a patient via intra-arterial, subcutaneous, intradermal, intratumoral, intranodial, intramedullary, intramuscular, intravenous (i.v.) injection or intraperitoneal administration. In one aspect, the T cell composition of the invention is administered to a patient by intradermal or subcutaneous injection. In one aspect, a CAR-expressing cell (e.g., T cell or NK cell) composition of the invention is administered by intravenous injection. Compositions of cells expressing CAR (eg, T cells or NK cells) can be injected directly into tumors, lymph nodes, or sites of infection.

In a particular exemplary aspect, the individual can undergo leukapheresis, wherein the leukocytes are collected, enriched, or depleted ex vivo to select and/or isolate related cells, such as immune effector cells (eg, T cells or NK cells). Such immune effector cell (e.g., T cell or NK cell) isolates can be amplified and processed by methods known in the art such that one or more of the CAR constructs of the present invention can be introduced, thereby producing the performance of the present invention. CAR cells (example Such as CAR T cells or NK cells expressing CAR). Individuals in need can then undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In certain aspects, the individual is subjected to an infusion of an expanded CAR-expressing cell (e.g., CAR T cell or NK cell) of the present invention, either at the same time as or after transplantation. In another aspect, the expanded CAR-expressing cells (eg, CAR T cells or NK cells) are administered before or after surgery.

In embodiments, lymphocyte depletion is performed on an individual, for example, prior to administration of one or more cells that exhibit a CAR as described herein, such as a BCMA-binding CAR as described herein. In an embodiment, depletion of lymphocytes comprises administering one or more of melphalan, dexamethasone, cyclophosphamide, and fludarabine.

The dosage of the above-described treatment to be administered to the patient will vary depending on the exact nature of the condition being treated and the recipient of the treatment. The dosage for human administration can be scaled according to practices accepted in the art. The dose of CAMPATH will, for example, be in the range of from 1 to about 100 mg for an adult patient, usually administered daily for a period of from 1 to 30 days. A preferred daily dose is from 1 to 10 mg per day, although in some cases larger doses up to 40 mg per day may be used (described in U.S. Patent No. 6,120,766).

In one embodiment, the CAR is introduced into an immune effector cell (eg, a T cell or an NK cell), for example, using in vitro transcription, and the individual (eg, a human) receives an initial of the CAR immune effector cell (eg, T cell or NK cell) of the present invention. Administration, and one or more subsequent administrations of the CAR immune effector cells (eg, T cells or NK cells) of the invention, wherein one or more subsequent administrations are less than 15 days after the previous administration, eg, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 days of administration. In one embodiment, more than one administration of a CAR immune effector cell (eg, a T cell or an NK cell) of the invention is administered to an individual (eg, a human) per week, eg, 2, 3, or 4 times per week. Administration of CAR immune effector cells (eg T cells or NK cells). In one embodiment, an individual (eg, a human subject) receives more than one CAR immune effector cell (eg, T cell or NK cell) administration per week (eg, 2, 3, or 4 weekly administrations) (also referred to herein as For the cycle), do not vote for CAR immunization for the following week Cells (eg, T cells or NK cells) should be administered to the individual for additional administration of one or more CAR immune effector cells (eg, T cells or NK cells) (eg, more than once per week of CAR immune effector cells (eg, T cells) Or NK cells)). In another embodiment, an individual (eg, a human individual) receives more than one CAR immune effector cell (eg, T cell or NK cell) cycle, and the time between cycles is less than 10, 9, 8, 7, 6, 5, 4 or 3 days. In one embodiment, CAR immune effector cells (eg, T cells or NK cells) are administered every other day, three times a week. In one embodiment, the CAR immune effector cells (eg, T cells or NK cells) of the invention are administered for at least two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, or more than eight weeks.

In one aspect, a lentiviral vector, such as a lentivirus, is used to produce cells expressing BCMA CAR (eg, BCMA CART or NK cells expressing BCMA CAR). Cells expressing CAR that are produced in this way (eg, CART or NK cells expressing CAR) will have stable CAR performance.

In one aspect, a viral vector, such as CART, is produced using a viral vector, such as a gamma retroviral vector, such as the gamma retroviral vector described herein. CART produced using such vectors can have a stable CAR performance.

In one aspect, after transduction, cells expressing CAR (eg, CART or NK cells expressing CAR) transiently exhibit CAR carriers for 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 days. Short-term performance of CAR can be achieved by RNA CAR vector delivery. In one aspect, CAR RNA is transduced into cells such as T cells or NK cells by electroporation.

Potential problems that may arise in patients treated with cells that transiently express CAR (eg, CART or CAR-expressing NK cells), particularly those that use CAR-bearing scFv-presenting CAR-like cells, such as CART or CAR-expressing NK cells Systemic allergic reaction after secondary treatment.

Without being bound by this theory, this kind of allergic reaction can be caused by the production of humoral anti-CAR It should be caused by a patient (ie, an anti-CAR antibody with an anti-IgE isotype). When there is an interruption in antigen exposure from ten days to fourteen days, it is considered that the antibody-producing cells of the patient undergo a categorical conversion of the IgG isotype (without causing a systemic allergic reaction) to the IgE isotype.

If the patient is at high risk of developing an anti-CAR antibody response (such as a response by RNA transduction) during transient CAR therapy, the disruption of infusion of cells expressing CAR (eg, CART or CAR-expressing NK cells) should not be sustained More than ten days to fourteen days.

Instance

The invention is further described in detail with reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting. Therefore, the present invention should not be construed as being limited to the following examples, but should be construed as covering any and all variations that are apparent from the teachings provided herein.

Without further elaboration, the skilled artisan can make and utilize the compositions of the present invention and practice the claimed methods using the foregoing description and the following illustrative examples. The following examples are illustrative of various aspects of the invention and are not to be construed as limiting the remainder of the invention in any way.

Example 1: BCMA behaves in myeloma cell lines and major samples Analysis of BCMA expression in myeloma cell lines by quantitative PCR

Sixteen cell lines of human cancer were screened for BCMA RNA expression by quantitative RT-PCR. RNA was extracted using the RNAqueos-4 PCR kit (Ambion, AM-1914), and cDNA was synthesized using RT-qPCR iScript Reverse Transcription Supermix (BioRad, 170-8841). Relative BCMA cDNA copies were quantified by relative qPCR (qPCR) using ABI TaqMan BCMA specific primers and probe sets (ABI, Hs03045080, lot number: 1139777); TaqMan GUSB (ABI, Hs99999908_M1, lot number: 1093869) primers and probe sets Used for calibration.

Analysis of qPCR showed that all tested MM cell lines (U266, NCI H929 and RPMI 8226) exhibited BCMA. Also in BJAB and LCL cells (B cell lymphoblastoid cells) BCMA was detected in cell lines and in CEM cells (T-type lymphoblastic cell lines). None of the other non-MM cell lines showed detectable BCMA performance. This RNA analysis supports the detection of proteins by flow cytometry to select positively expressed BCMA cell lines for evaluation. Detailed results are provided in Figure 1A.

A comparison by RNA analysis from BCMA expression in plasma cells of patients and in different multiple myeloma samples is provided in Figure IB.

Analysis of BCMA expression in multiple myeloma cell lines and major samples by flow cytometry

Multiple myeloma (MM) cell line U266, NCI H929 or RPMI 8226 from MM patients (PB or BM) or primary samples were stained with human BCMA/TNFRSF17 phycoerythrin affinity purified PAb, goat IgG (R&D, FAB193P) . The main samples from patients with multiple myeloma also used Live/dead dyes (Life Technologies, L34960), CD45-BV421 (Biolegend, 304032), CD38-APCeF780 (eBioscience 47-0389-42), CD138-APC (eBioscience 17-1389). -42), CD19-PECY7 (eBioscience, E10328-1632), lambda chain-PerCP-eF710 (eBioscience 46-9990-42) and kappa light chain (eBioscience, 11-9970-42) staining. Data from stained samples were collected using a BD Fortessa cell counter. Flow cytology analysis was performed using Flowjo v10 (Tree Star Inc).

As shown in Figures 2A, 2B and 2C, BCMA was detected on the surface of all three MM cell lines. In addition, BCMA was uniformly expressed on most pure (kappa or lambda-restricted) plasma cells in most of the MM patients analyzed (9 out of 10) (Figures 2D and 2E). These results provide strong support for the association of BCMA as a target in MM.

Flow cytological analysis of BCMA expression in normal peripheral blood cells, CD3/CD28 expanded T cells and bone marrow stem cells

To exclude possible off-target performance of BCMA in normal tissues and on T cells, two bone marrow (BM) and peripheral blood (PB) from voluntary healthy donors were assessed by flow cytometry. BCMA performance in the sample. Monocytes were obtained by Gracoll-Paque (GE healthcare) gradient separation. BM cells were then labeled with Live/dead dye (Life Technologies, L34960) followed by CD34-APC (eBioscience, 17-0349-42), CD38-PECY7 (eBioscience, 25-0389-42), human hematopoietic lineage markers Mixture - FITC (eBioscience, 22-7778-72), CD45RA-APC-eF780 (eBioscience, 47-0458-42), CD90-PerCPCy5.5 (eBioscience, 45-0909-41), CD10-BV421 (Biolegend, 312218) And BCMA-PE (R&D, FAB193P) single antibody staining. Fresh PB cells were stained at baseline and after stimulation and expansion with CD3/CD28 beads. PB was used for CD14-V500 (BD, 561391), CD45-BV421 (Biolegend, 304032), CD3-AF700 (56-0038-42), CD19-PECY7 (eBioscience, E10328-1632), BCMA-PE (R&D, Single antibody staining of FAB193P). Cells were washed twice and staining data were obtained in a BD Fortessa cell counter. Flow cytology analysis was performed by using FlowJo v10 (Tree Star Inc).

No evidence of BCMA performance was observed on the PBMC. Importantly, T cells remained BCMA negative during expansion (Fig. 3A). Analysis of different stem cell subsets in BM revealed that BCMA did not be expressed on immature lineage-negative CD34-positive stem cells. In detail, common lymphocyte progenitor cells and hematopoietic stem cells were negative (Fig. 3B).

Analysis of BCMA expression in normal tissues by immunohistochemistry

Three commercially available antibodies (Novus, Sigma) for immunohistochemistry were selected and titrated in paraffin-fixed normal spleen tissue. Tissue microarrays (TMA) comprising 27 healthy human tissues were stained by immunohistochemistry.

All three antibodies showed positive staining on normal plasma cells in lymph nodes, spleen and tonsils, while the normal lung, pancreas and thyroid gland tested were negative. Staining is observed in the following organs, which are likely to be non-specific due to the availability of multiple antibodies: stomach, salivary gland, kidney, adrenal gland, cerebellum, heart and appendix. Selected results are shown in Figures 4A-4E and summarized in Table 11.

These results led to further analysis of performance, particularly the use of RNAscope in situ hybridization to confirm the lack of BCMA performance in these tissues. Selected results are shown in Figure 4F.

Example 2: In Vitro Evaluation of CAR Containing Humanized Anti-BCMA scFv BCMA CAR construct produced from humanized mouse anti-BCMA antibody

Designing four different anti-BCMA CAR constructs using the VL and VH sequences disclosed in PCT Publication WO 2012/163805, the contents of which are incorporated herein in its entirety by reference. body. To generate anti-BCMA CAR, the VH and VL sequences were synthesized and ligated with the [Gly-Gly-Gly-Gly-Ser] x 4 linker (SEQ ID NO: 27) to generate two single-chain variable fragments (scFv), Wherein VH is before VL (H2L, SEQ ID NO: 255) or VL is before VH (L2H, SEQ ID NO: 257). The CD8 leader sequence was also synthesized and fused to the 5' end of each scFv with a BamHI site. The restriction sites of XbaI and BspE1 were included at the 5' and 3' ends, respectively, to facilitate the selection of the CD8 leader sequence-scFv into the pTRPE lentiviral vector containing the hinge and the CD8TM region with 4-1BB and CD3z cytoplasmic domains. . Two separate CAR backbone constructs were used for selection, one containing the human CD8 hinge and the other containing the human IgG4 hinge to generate the four anti-BCMA CAR constructs schematically shown in Figure 5, designated pBCMA 1 , pBCMA 2, pBCMA 3 and pBCMA 4. To generate infectious lentiviral vector supernatants, 293-T cells were plated with the following plasmids: pTRP-VSV-G (encoded vesicular stomatitis virus (VSV-G) envelope), pTRP gag/pol (coded gag and Pol) and pTRP-Rev were transfected with lipofectamine 2000 (Invitrogen) using any of the four BCMA CAR constructs.

The nucleic acid sequences of the humanized anti-BCMA scFv (H2L, such as pBCMA 2 and pBCMA 4) of VH before VL are as follows: (SEQ ID NO: 272)

The corresponding amino acid sequence of the humanized anti-BCMA scFv of Vh before VL (H2L, such as pBCMA 2 and pBCMA 4) is as follows: (SEQ ID NO: 271)

The nucleic acid sequences of the humanized anti-BCMA scFv (L2H, such as pBCMA1 and pBCMA3) of VL before VH are as follows: (SEQ ID NO: 274)

The corresponding amino acid sequence of the humanized anti-BCMA scFv of VL before VH (L2H, such as pBCMA1 and pBCMA3) is as follows: (SEQ ID NO: 273)

These pBCMA-CARs containing the humanized anti-BCMA scFv were utilized in the experiments detailed below and in Example 3.

Effective performance of BCMA-CAR on T cells

Freshly isolated human T cells from healthy donors were transduced with lentiviral vector supernatants encoding pBCMA 1 to 4 CAR and anti-BCMA CAR performance was assessed by flow cytometry. Briefly, T cells were cultured in RPMI 1640 medium with 10% FBS and stimulated with anti-CD3/anti-CD28 Dynabead (Invitrogen). T cells were transfected with four different pBCMA CAR lentiviral vector supernatants 24 hours after stimulation. guide. T cells transduced with anti-mesothelin CAR (SS1) vector were used as positive controls. Simulated transduced T cells (NTD) were used as negative controls. 4-6 days after lentiviral transduction, T cells were stained with biotinylated protein L antibody, followed by streptavidin FITC (BD Biosciences) or biotin goat-anti-mouse, and by flow cell volume The CAR performance was assessed by FACS Calibur (BD). Flow cytology analysis was performed by using Flowjo (Tree Star Inc).

After transduction, pBCMA CAR was observed to be effectively expressed on the cell surface of transduced T cells, as shown in Figure 6.

Cytokine production from anti-BCMA CAR T cells (BCMA CART)

K562 cells ectopically expressing human BCMA (K562-BCMA) were generated by lentiviral transduction using a vector provided by Gene Copoeia followed by puromycin selection. The cytotoxicity and production of cytokines from pBCMA 1-4 CAR-transduced T cells were evaluated in vitro using K562-BCMA-specific target cells. Anti-pBCMA CAR T cells or control T cells were expanded until the end of log phase growth, and subsequently with K562-BCMA-specific target cells, K562-mesothelin as a positive control, or as a negative control Target cells were co-cultured with target cells at a 3:1 ratio for 16 hours. Culture supernatants were harvested and IFN-[gamma] and IL-2 concentrations were measured by specific ELISA according to the manufacturer's instructions (R&D).

As shown in Figures 7A and 7B, T cells expressing all four anti-pBCMA CARs produced similar levels of IFN-γ and IL when co-cultured with target cells expressing BCMA but not with BCMA negative target cells. -2.

Cytotoxic activity of BCMA CART on myeloma cell lines

The ability of pBCMA CAR T cells to kill target cells expressing BCMA was assessed using a ⁵¹ Cr release assay. Briefly, target cells were labeled with MM ⁵¹ Cr (sodium dichromate), and washed with effector pBCMA CAR T cells at different effector / target ratio of co-culture. Supernatants were collected at 4 hours and placed in 96-well Lumaplate (Perkin Elmer). The amount of ⁵¹ Cr released from the labeled target cells was measured on a liquid scintillation counter (MicroBeta trilux, Perkin Elmer). Target cells incubated in medium alone or with 1% SDS were used to determine spontaneous (S) or maximum (M) ⁵¹ Cr release. The specific percentage of dissolution was calculated as follows: 100 x (cpm experimental release - cpm S release) / (cpm M release - cpm S release).

As shown in Figures 8A, 8B, 8C and 8D, all four pBCMA-CAR transduced T cells were able to induce lysis of K562-BCMA cells and multiple myeloma cell lines expressing BCMA, with almost BCMA negative cell lines. Inactive. The pBCMA-CAR (pBCMA 3 and 4) with CD8 hinge showed greater cytotoxicity than the pBCMA CAR containing the IgG4 hinge, indicating that the hinge is an important factor in the CAR design for optimal function.

Example 3: In vivo evaluation of pBCMA-CART for multiple myeloma

Based on the enhanced in vitro data supporting the functional enhancement of pBCMA 3 and pBCMA 4 CAR-modified T cells, the anti-tumor activity of these CARTs was evaluated using a RPMI 8226 cell line in a preclinical animal model of multiple myeloma. RPMI 8226 cells were engineered to express clondal green luciferase (CB-G Luc ⁺ ) to track tumor progression by bioluminescence in vivo imaging (IVIS) and Living Image software (Perkin Elmer). Four weeks after injection of CB-G Luc ⁺ , RPMI 8226 cells were injected IV in NSG recipients, and IV injections showed pBCMA 3 CAR, pBCMA 4 CAR, CD19 CAR (FMC63 with human CD8 hinge, 4-1BB and CD3z cytoplasmic domains). T cells with anti-CD19 scFv) or SS1 CAR (SS1 anti-mesothelin scFv with human CD8 hinge, 4-1BB and CD3z cytoplasmic domains), and tumor burden assessed by optical imaging and by clinical signs of disease (Table 12 below). The scores were as follows: 1- no clinical symptoms; 2 - micro gait changes / smaller tumor mass; 3 - reduced activity / tumor mass / still walking; 4 - hind limb paralysis / large tumor mass / endpoint; and 5- Complete limb paralysis.

As shown in Figures 9A and 9B, T cells expressing pBCMA-3 and pBCMA 4 CAR induced tumor burden in mice bearing RPMI 8226 compared to control T cells targeting mesothelin (designated SS1) or CD19. Significantly reduced, as well as increased clinical disease activity.

The experiments described in Examples 2 and 3 provide the rationale for identifying human scFv binding domains for the additional CAR constructs described and evaluated in Examples 4-7.

Example 4: Production of human anti-BCMA CAR constructs

Human BCMA-specific scFvs for CAR constructs were identified by 3 rounds of bead-based Bio-BCMA panning. In arm 1, SBAL-1Sk phage library (8.4E13) was used. In arm 2, SBAL-3Sk (G1+G2+G4+G5) phage library (1E13) was used. Phage lysates were screened for BCMA reactivity by ELISA. Identify 319 positive collisions representing 135 unique sequences. The phage sequence is transformed into a soluble scFv that can be expressed in E. coli. E. coli lysates were then screened by ELISA and the extracellular mass was screened by FACs. 15 collisions were identified by these FACs analysis. Subsequently, the scFv was purified from E. coli and the purified scFv was tested by FACs. Confirmed 15 scFvs, and named BCMA-1, BCMA-2, BCMA-3, BCMA-4, BCMA-5, BCMA-6, BCMA-7, BCMA-8, BCMA-9, BCMA-10, BCMA- 11. BCMA-12, BCMA-13, BCMA-14 and BCMA-15. The sequence of the human anti-BCMA scFv fragment (SEQ ID NOS: 39-52) is provided in Table 1 (and the name is provided in Table 2). The human scFv fragment (SEQ ID NO: 39-52) was used in combination with the additional sequences shown in the examples (eg, leader sequence, CD8 hinge, CD8 transmembrane, 4-1BB intracellular domain, CD27 intracellular domain, CD28 cell) Combination of an internal domain, an ICOS domain, a CD3ζ domain (mutation), a human CD3ζ domain, an IgG4 hinge, a Gly Ser sequence, and/or a poly(A) sequence to generate a complete BCMA CAR construct (SEQ ID NO: 99- 113).

The CAR scFv fragment was then cloned into a lentiviral vector in a single coding frame and used for expression (SEQ ID NO: 11) using the EF1 alpha promoter to form a full-length CAR construct.

The amino acid and nucleic acid sequences of the BCMA scFv domain and the BCMA CAR molecule are provided in Table 1 of the Examples. Table 2 in the examples indicates the nickname of the BCMA CAR construct relative to the DNA ID number also listed in Table 1.

The VH and VL sequences from PCT Publication No. WO 2012/0163805, the contents of which are hereby incorporated by reference in its entirety, are also used to produce additional tools BCMA CAR constructs, and based on the pBCMA3 and pBCMA4 CARs described in Examples 2 and 3. The result. A schematic of the tool BCMA constructs (BCMA-3NP and BCMA-4NP) is shown in Figure 10A. The orientations of the VH and VL chains of the two constructs are different (Fig. 10B). The tool BCMA CAR construct and its corresponding DNA ID are shown below.

Other BCMA CAR constructs were generated using the VH and VL sequences found in Table 16 of the Examples. An exemplary scFv domain comprising the VH and VL domains and a linker sequence and an amino acid sequence of the full length CAR are also found in Table 16.

Generation of BCMA CAR structures and calculation of viral price

Lentiviral supernatants were generated from 15 BCMA-specific CAR constructs (Table 2) obtained from scFv phage screens. Two lentiviral supernatants of BCMA tool CAR constructs BCMA-3NP and BCMA-4NP were also generated. The tool building system described in this example is based on the pBCMA3 and pBCMA4 constructs previously described in Examples 2 and 3.

To generate lentiviral supernatants, LentiX-293T cells were seeded on day 0 and transfected on day 1 with Lipofectamine 2000 transfection reagent (Life Technologies). For each construct, the plastid DNA used is: pRSV.REV (Rev representation plastid), pMDLg/p.RRE (Gag/Pol for plastids), pVSV-G (VSV glycoprotein for plastids) and CAR transfer vector. The transfection mixture was replaced with fresh medium on day 2 and virus supernatants were collected on days 3 and 4.

The viral supernatant was concentrated using Lenti-X Concentrator reagent (Clontech) and the resulting pellet was resuspended in growth medium at an initial volume of 1/10 to 1/100. The concentrated virus supernatant was aliquoted and stored at -80 °C. The calculation of viral power price was determined by transducing SupT1 cells and assessing CAR performance. SupT1 cells were transduced with virus supernatant at 3 fold serial dilutions on day 1 at a starting concentration of 1:300. CAR performance was assessed on day 5 with BCMA-Fc antigen (R&D Systems) and Biotin-Protein L reagent (GenScript). Calculate the viral power price according to the following formula: (% CAR+) × (# SupT1 cells) / (virus amount (ml)) × (diluted)

The mean viral power price was calculated from the dilution point in the linear range between 1% and 20% CAR positive. The calculation of the price of the BCMA CAR pure system is shown in Table 4.

Although all BCMA CAR pure lines are detected with BCMA-Fc antigen, for many For the pure lines (BCMA-1, BCMA-6, BCMA-11), the detection with Biotin-protein L was weak. The force price based on BCMA-Fc detection was used to calculate the MOI of transduction in primary T cells. The BCMA CAR construct containing the human anti-BCMA scFv described in this example was used throughout the experiments of Examples 4 and 5. The tool BCMA CAR constructs described in this example were also used in the experiments described in Examples 5, 6 and 7.

Example 5: In vitro characterization of tool BCMA CAR constructs JNL Reporter Gene Analysis of Tools BCMA CAR Constructs

Jurkat-NFAT-luciferase (JNL) cells transduced with the tool BCMA CAR construct were evaluated for response to activation of the target cell line expressing BCMA. On day 0, JNL cells were transduced with BCMA-3NP and BCMA-4NP. The virus concentration was adjusted to MOI 3 and incubated overnight. On day 6 after transduction, NBMA CAR-transduced JNL cells were incubated with target cells at a ratio of target (E:T) in the range of 0 to 27. The target cell lines were: K562 (BCMA negative control), and BCMA positive multiple myeloma cell lines NCI-H929, KMS26 and RPMI 8226. On day 7, JNL activation was measured using Bright-Glo prime (Promega). On day 6, CAR performance on transduced JNL was assessed using BCMA-Fc antigen (Table 5). This reporter gene analysis confirmed that the CAR pure line of the targeting tool BCMA was activated in a target-specific manner (Figure 11).

The progress of primary T cell isolation, CAR transduction and activation of the tool targeting BCMA is shown in Table 6. On day 0, healthy human donor PBMC (Novartis Employee Blood Donor program) was isolated from whole blood by Ficoll extraction, and T cells were isolated from PBMC by negative selection using Pan T Cell Isolation Kit II (Miltenyi Biotec). Isolated T cells with Dynabeads Human T-Expander CD3/CD28 beads (Life Technologies) stimulated overnight at a 3:1 ratio of beads to cells. T cells were also stained to assess the relative amounts of CD4+ and CD8+ cells (Figure 12).

On day 1, T cells were transduced with BCMA-3NP and BCMA-4NP. The virus concentration was adjusted to MOI 5 and incubated overnight. On day 11, the transduced CART cells were removed from the beads and aliquoted in 90% FBS, 10% DMSO. After transduction and amplification, T cells were again stained to assess the relative amounts of CD4+ and CD8+ cells. In addition, CAR performance was evaluated using BCMA-Fc antigen (Fig. 13).

BCMA proliferation analysis

The assessment responded to BCMA CART cell proliferation in response to target cells expressing BCMA. CART cells were thawed on day 0 and grown overnight for recovery. On day 1, CART cells were labeled with CellTrace CFSE (Life Technologies) and incubated with irradiated target cells at a 1:1 E:T ratio. Dynabeads Human T-Expander CD3/CD28 beads with a 3:1 bead to cell ratio were included as positive controls. On day 5, the CFSE content in CART cells was measured (Fig. 14). In addition, CART cells were stained with CD3, CD4, CD8, and BCMA-Fc antigens and measured by flow cytometry relative to CountBright Absolute Count Beads (Life Technologies) to determine relative cell numbers (Figure 15). . For BCMA-3NP and BCMA-4NP, specific proliferation was observed in response to BCMA.

BCMA CART luciferase cell killing assay

The assessment responded to BCMA CART cell killing in response to target cells expressing BCMA. CART cells were thawed on day 0 and grown overnight for recovery. On day 1, CART cells were incubated with KMS11-luciferase or MM1-S-luciferase target cells expressing BCMA at an E:T ratio ranging from 0 to 20. On day 2, the luciferase signal loss caused by cell killing was measured by mass using Bright-Glo and the specific lysis was calculated according to the following formula: specific lysis (%) = 100 - (sample luminescence / average maximum luminescence) × 100

The results of cell killing are shown in Figure 16, confirming that the tool CART pure line has a specific killing response.

BCMA CART CFSE Cell Kill Analysis

The assessment responded to BCMA CART cell killing in response to target cells expressing BCMA. CART cells were thawed on day 0 and grown overnight for recovery. On day 1, target cells were labeled with CellTrace CFSE (Life Technologies) and incubated with BCMA CART cells at an E:T ratio ranging from 0 to 10. The target cell lines were: K562-BCMA (engineered to stably express BCMA), K562 (parent cell line) and BCMA positive multiple myeloma cell lines NCI-H929, KMS26 and RPMI 8226. On day 2, CFSE-positive cell loss from cell killing was measured by flow cytometry relative to CountBright absolute counting beads (Life Technologies) to determine relative cell numbers (Figure 17). Specific cell killing was observed for both BCMA-3NP and BCMA-4NP.

Based on the in vitro characterization of the BCMA tool CAR pure line, BCMA-3NP and BCMA-4NP were selected for in vivo evaluation in the KMS11-luciferase disseminated tumor model. UTD (untransduced) T cells were selected as a negative control. The results from in vivo characterization are further described in Example 5.

Example 6: In vitro characterization of BCMA CART

The experiments described in this example utilized a CAR construct containing the human anti-BCMA scFv from Table 1 and a tool BCMA CAR construct.

JNL Reporter Gene Analysis of BCMA AR Constructs

Jurkat-NFAT-luciferase (JNL) cells transduced with BCMA CAR constructs were evaluated for response to activation of target cells expressing BCMA. Small scale viral supernatant samples were generated in HEK293T cells to transduce JNL cells. On day 3 after transduction, BCMA CAR-transduced JNL cells were incubated with target cells at a 6:1 effect to target (E:T) ratio. The target cell lines were: K562-BCMA (engineered to stably express BCMA), K562 (parent cell line) and BCMA-positive multiple myeloma cell lines NCI-H929 and RPMI 8226. On day 4, JNL activation was measured using Bright-Glo prime (Promega). On day 7, CAR performance on transduced JNL was assessed using BCMA-Fc antigen and biotin-protein L reagent (Table 7). This reporter gene analysis confirmed that several CAR pure lines targeting BCMA were activated in a target-specific manner (Figure 18).

JNL activity is not associated with % CAR performance. Based on BCMA-specific activation, the following BCMA CAR pure lines were selected for characterization in primary T cells: BCMA-1, BCMA-4, BCMA-5, BCMA-7, BCMA-8, BCMA-10, BCMA-12, BCMA-13 , BCMA-14 and BCMA-15. Based on the observed non-specific activation, BCMA-6 was selected as a negative control. Based on the observed lack of activity, BCMA-9 was also selected as a negative control.

BCMA CAR transduction of primary human T cells

The progress of primary T cell isolation, CAR transduction and activation of the tool targeting BCMA is shown in Table 8. On day 0, healthy human donor PBMC (Novartis Employee Blood Donor program) was isolated from whole blood by Ficoll extraction, and T cells were isolated from PBMC by negative selection using Pan T Cell Isolation Kit II (Miltenyi Biotec). Isolated T cells were stimulated overnight with Dynabeads Human T-Expander CD3/CD28 beads (Life Technologies) at a ratio of beads to cells of 3:1. T cells were also stained to assess the relative amounts of CD4+ and CD8+ cells (Figure 19).

On day 1, T cells were transduced with the following BCMA CAR pure lines: BCMA-1, BCMA-4, BCMA-5, BCMA-6, BCMA-7, BCMA-8, BCMA-9, BCMA-10, BCMA-12 BCMA-13, BCMA-14, BCMA-15, BCMA-3NP and BCMA-4NP. Virus concentrations were adjusted to MOI 5 (Table 9) and grown overnight.

On day 11, the transduced CART cells were removed from the beads and aliquoted in 90% FBS, 10% DMSO. After transduction and amplification, T cells were again stained to assess the relative amounts of CD4+ and CD8+ cells. In addition, CAR expression was evaluated using BCMA-Fc antigen (Fig. 20).

BCMA CART proliferation analysis

The assessment responded to BCMA CART cell proliferation in response to target cells expressing BCMA. CART cells were thawed on day 0 and grown overnight for recovery. On day 1, CART cells were labeled with CellTrace CFSE (Life Technologies) and incubated with irradiated target cells at a 1:1 E:T ratio. On day 6, CFSE levels in CART cells were measured (Figure 21).

In addition, CART cells were stained with CD3, CD4, and CD8 and measured by flow cytometry versus CountBright Absolute Count Beads (Life Technologies) to determine relative cell numbers (Figures 22A, 22B, and 22C). Specific proliferation in response to BCMA was observed for the following CART pure lines: BCMA-4, BCMA-10, BCMA-12, BCMA-13, BCMA-14 and BCMA-15.

BCMA CART Kill Analysis

The assessment responded to BCMA CART cell killing in response to target cells expressing BCMA. CART cells were thawed on day 0 and grown overnight for recovery. Day 1, CART cells and tables The KMS11-luciferase target cells of BCMA are now incubated together at an E:T ratio in the range of 0 to 10. On day 2, the luciferase signal loss caused by cell killing was measured by mass using Bright-Glo and the specific lysis was calculated according to the following formula: specific lysis (%) = 100 - (sample luminescence / average maximum luminescence) × 100

The results of the cell kill assay are shown in Figure 23A, and each BCMA CAR construct was compared to BCMA-3NP and BCMA-4NP. These results confirmed that several CART pure lines (expressing human anti-BCMA scFv) had greater killing responses than the control BCMA-3NP and BCMA-4NP constructs. Results from selected candidate BCMA CARs (BCMA-4, BCMA-9, BCMA-10, BCMA-13, and BCMA-15) are presented graphically in Figure 23B to compare killing ability between candidate CARs. Untransduced T cells and T cells transduced with the BCMA-4NP construct were used as negative and positive controls, respectively. For each CART, the percentage of cell killing of selected BCMA CART pure lines (BCMA-4, BCMA-9, BCMA-10, BCMA-13, and BCMA-15) was corrected relative to CAR performance % and is presented in Figure 23C. The results show that the BCMA-4, BCMA-9, BCMA-10, BCMA-13 and BCMA-15 CART pure lines all have cell killing ability similar to BCMA-4NP.

An overview of the in vitro analysis of the above BCMA CART pure lines is shown in Table 10. Based on in vitro characterization of BCMA CART pure lines, BCMA-4, BCMA-10, BCMA-13 and BCMA-15 were selected for in vivo evaluation in the KMS11-luciferase disseminated tumor model, as in Example 7. Said. BCMA-4NP was selected as a positive control. BCMA-9 and UTD (untransduced) were selected as negative controls.

Example 7: In vitro characterization of BCMA CART

KMS-11 is a human multiple myeloma cell line derived from IgGκ pleural effusion, and can be grown as a xenograft in immunocompromised mice. This xenograft will mimic a disease in the bone marrow as seen in humans, establishing a model in which the efficacy of the test therapy on multiple myeloma in the bone is tested. These mice can be used to test the efficacy of chimeric antigen receptor (CAR) T cells specific for cell markers found on plasma cells and multiple myeloma cells, such as B cell mature antigen (BCMA). The genetic markers reported KMS-11 cells by the firefly luciferase and used to test the specificity of having ^BCMA NOD.Cg- Prkdc ^scid Il2rg tm1wjl / orthotopic model SzJ (NSG) mice of multiple myeloma in The efficacy of CAR T cells.

BCMA expression was tested on KMS-11 cells and these cells were used in in vitro assays to view BCMA-specific CAR T cell recognition and ability to respond to targets. The growth and growth of KMS-11 cells in vivo when implanted intravenously via the tail vein is mainly limited to bone marrow. One week after implantation of the tumor cells, the disease is completely displaced to the bone and begins to grow at an exponential rate. Untreated mice 5-6 weeks after tumor implantation will begin to show clinical signs and hind limb paralysis. The tool BCMA CAR T cells were first tested in efficacy studies in this model to determine if the model is a suitable in vivo model for testing the efficacy and anti-tumor activity of BCMA CAR T cells. Thereafter, the primary BCMA scFv from in vitro selection was tested in this in vivo model and is now confirmed in repeated efficacy studies.

Materials and Methods:

KMS-11 cell line: KMS-11 human multiple myeloma cell line was produced from pleural effusion in patients with multiple myeloma. The cells were then labeled with firefly luciferase. These suspension cells were grown in RPMI supplemented with 10% heat-inactivated fetal bovine serum.

Mice: 6 weeks old ^{NSG (NOD.Cg- Prkdc scid Il2rg tm1Wjl /} SzJ) mice from Jackson Laboratory (storage number 005,557) received. Animals were acclimatized to the new environment in the Novartis NIBRI animal facility for at least 3 days prior to the experiment. Animals are disposed of according to Novartis ACUC regulations and guidelines.

Tumor transplantation: KMS-11-luc cells were grown and expanded in vitro in RPMI supplemented with 10% heat-inactivated fetal bovine serum. The cells were then transferred to a 15 ml conical tube and washed twice with cold sterile PBS. Next, KMS-11-luc cells were counted and at a concentration of 10 × 10 ⁶ cells per ml of PBS and resuspended. The cells were placed on ice and immediately (into one hour) implanted into the mice. KMS-11 cells were injected intravenously via the tail vein in a volume of 100 μl, for a total of 1 × 10 ⁶ cells per mouse.

CAR T cells were administered: 5 x 10 ⁶ T cells were administered to mice 7-8 days after tumor implantation. The cells were decomposed in the middle of a 37 ° C water bath and then completely thawed by adding 1 ml of cold sterile PBS to the tubes containing the cells. The thawed cells were transferred to a 15 ml falcon tube and adjusted to a final volume of 10 ml with PBS. The cells were washed twice at 1000 rpm for 10 minutes each, followed by counting on a hemocytometer. CAR T cells were corrected for CAR transduction such that each group had the same percentage of CAR ⁺ T cells. A total of 5 x 10 ⁶ cells were then resuspended at a concentration of 50 x 10 ⁶ cells per ml of cold PBS and stored on ice until given to the mice. Mice were intravenously injected with 100 μl of CAR T cells via the tail vein at a dose of 5 × 10 ⁶ T cells per mouse.

Five to seven mice per group were treated with 100 μl of PBS alone (PBS), untransduced T cells (mock), tools BCMA CAR T cells (BCMA-3NP or BCMA-4NP) or novel BCMA CAR T cells (BCMA- 4. BCMA-9, BCMA-10, BCMA-13, BCMA-15). T cells were all prepared simultaneously from the same human donor.

Animal Monitoring: The health of the mice was monitored daily, including weekly measurements of body weight twice. The percent change in body weight was calculated as (BW _{now -} BW _initial ) / (BW _initial ) x 100%. Tumor burden was monitored twice weekly by bioluminescence imaging. Mice were injected intraperitoneally with D-luciferin 10 minutes prior to anesthesia and mice were imaged with Xenogen. The disease burden was calculated by calculating the bioluminescence (photons/second) of the tumor cells.

Bioluminescence analysis: The treatment/control percentage (T/C) value is calculated using the following formula: if ΔT 0, then T/C%=100×△T/△C; if ΔT<0, return %=100×ΔT/T _initial ; wherein T=the average bioluminescence of the drug treatment group on the last day of the study; T _Initial = mean bioluminescence at the start of dosing in the drug-treated group; ΔT = average bioluminescence in the drug-treated group on the last day of the study - average bioluminescence at the start of dosing in the drug-treated group; C = control at the end of the study The average bioluminescence of one day; and ΔC = the average bioluminescence of the control group on the last day of the study - the average bioluminescence of the control group on the day of the start of dosing.

T/C values in the range of 100% to 42% are interpreted as having no or minimal antitumor activity; A 42% and >10% T/C value is interpreted as having anti-tumor activity or tumor growth inhibition. T/C value 10% or regression value -10% is explained as tumor stagnation. <-10% of the regression value is reported as subsided.

Peripheral blood FACS analysis: T cells in peripheral blood of mice were also monitored. Mice were bled weekly via the tail vein to EDTA coated tubes stored on ice. 10-20 μl of blood was piped from the tube to a 96-well plate on ice. Red blood cells were solubilized with ACK red blood cell lysis buffer (Life Technologies, Cat. No. A10492-01) and then washed twice with cold PBS. Cells were incubated with human and mouse Fc block Fc blocking cocktails (Miltenyi Biotec, Cat. No. 130-059-901 and 130-092-575) for 30 minutes, and then with anti-mouse CD11b antibody (BD Biosciences) , catalog number 557960), anti-human CD4 antibody (BD Biosciences, catalog number 563550), anti-human CD8 antibody (BD Biosciences, catalog number 560347) and BCMA-Fc antibody (R&D Systems, catalog number 193-BC-050) Then, Ig secondary (Jackson ImmunoResearch) was incubated together. Cells were fixed with 2% paraformaldehyde solution for 20 minutes, washed and stored in PBS + 2% FBS overnight, then analyzed on BD Fortessa, followed by further analysis using FlowJo FACS analysis software. Cells were analyzed to determine the number of CAR ⁺ CD4 ⁺ and CD8 ⁺ T cells per milliliter of blood in KMS-11-luc tumor-loaded NSG mice. The number of T cells in the blood is reported as the mean ± standard error of the mean (SEM).

result:

The anti-tumor activity of the BCMA CAR T cells (BCMA-3NP and BCMA4-NP) was evaluated and directly compared in the KMS-11 model of human multiple myeloma. After the tumor was implanted on day 0, the mice were randomized to the treatment group and treated with 5 x 10 ⁶ T cells intravenously on day 7. Monitor multiple myeloma disease burden and animal health until the animal reaches the end point. On day 14 after administration of CART cells (day 21 after tumor implantation), mice in all groups were sacrificed when the disease load in the control group approached maximum luminescence via imaging.

A clear difference in disease burden was seen between the control group and the group treated with either of the tool CAR T cells, with P < 0.01 on day 14 after administration of CAR T cells. tool Both BCMA CAR T cells demonstrated similar ability to control the growth of human multiple myeloma in NSG mice. The T/C% value of the mock-transduced T cell group was 212.13%, confirming that the mock-transduced T cells had no anti-tumor activity.

The ΔT/C% values of the BCMA-3NP and BCMA-4P groups were 1.10% and 2.17%, respectively, confirming tumor stagnation after treatment with the tool BCMA CAR T cells. Bioluminescence imaging results are shown in Figure 24. Baseline KMS-11 tumor growth kinetics in intravenously implanted NSG mice were confirmed by the PBS treated group that did not receive any T cells. The mock treatment group received untransduced T cells that underwent the same in vitro expansion process as CAR T cells. These cells were used as T cell controls to show non-specific responses of T cells in this tumor model. Both PBS and mock-transduced T cell treated groups demonstrated continuous tumor progression throughout the experiment. After two tools BCMA CAR T cells were injected 5 × 10 ⁶ T cells control disease progression.

The study of the novel scFv leader sequence was initiated after confirming that the KMS-11-luc model responded to targeting via BCMA CAR T cells. After implantation of the tumor, the mice were again randomized to the treatment group and treated with 5 x 10 ⁶ T cells intravenously on day 7. Monitor multiple myeloma disease burden and animal health until the animal reaches the end point. The mice in each group were sacrificed when the disease load in the group approached maximum luminescence via imaging.

A clear difference in disease burden was seen between the control group and some of the groups treated with BCMA CAR T cells. The tool BCMA CAR T cells (BMCA-4NP) did not control KMS-11 tumor growth as it has previously demonstrated. However, some novel BCMA CAR T cells demonstrate the level of efficacy of this change in this multiple myeloma model. The T/C% values calculated for each group at the endpoints showed tumor growth arrest in the BCMA-10 and BCMA-13 groups. The mock-transduced T cell group had a T/C% value of 61.56%, confirming that the mock-transduced T cells had minimal to no anti-tumor activity. The ΔT/C% value of the BCMA-4P group was 32.03%, confirming some minimal antitumor efficacy after treatment with the tool BCMA CAR T cells. The BCMA-10 and BCMA-13 groups showed tumor growth arrest with T/C values of 0.07% and 6.04%, respectively. BCMA-4 show initial control Tumor growth was made, but tumors began to grow in this group with only one dose of T cells administered to each group. Bioluminescence imaging results from the first experiment are shown in Figure 25A. Baseline KMS-11 tumor growth kinetics in intravenously implanted NSG mice were confirmed by the PBS treatment group that did not receive any T cells. The mock treatment group received untransduced T cells that underwent the same in vitro expansion process as CAR T cells. These cells were used as T cell controls to show non-specific responses of T cells in this tumor model. Both PBS and mock-transduced T cell treated groups demonstrated continuous tumor progression throughout the experiment. In the BCMA CART cell group, BCMA-4, BCMA-10, and BCMA-13 exhibited antitumor activity, while the tools BCMA CAR T cells (BCMA-4NP) and BCMA-9 and BCMA-15 did not exhibit antitumor efficacy. A second experiment was performed and the results of bioluminescence imaging are provided in Figure 25B. Mice receiving untransduced T cells exhibited baseline KMS-11 tumor growth kinetics. BCMA-4NP* indicates the results from the BCMA-4NP CART pure line in the first experiment. In the second experiment, BCMA-10, BCMA-13 and BCMA-15 exhibited robust antitumor activity.

In addition to monitoring disease burden via bioluminescence, the number of CAR ⁺ T cells in each group was also monitored via peripheral blood FACS analysis. The FACS results for this study are shown in Figure 26. The anti-tumor effect of KMS-11 tumors also showed CD4 ⁺ CAR ⁺ and CD8 ⁺ CAR ⁺ T cell expansion in peripheral blood. The BCMA-4, BCMA-10, and BCMA-13 groups exhibited CD4 ⁺ CAR ⁺ proliferation peaks between day 10 and day 20 after T cell treatment. These same groups also display long-term CD8 ⁺ CAR ⁺ T cell expansion.

The anti-tumor activity of novel BCMA CAR-transduced T cells was evaluated in an efficacy study in NSG mice bearing a xenograft model of human multiple myeloma. These studies demonstrate that the KMS-11-luc model reproduces human multiple myeloma in NSG mice and can be targeted by BCMA CAR T cells (Figure 24). This model was tested to be suitable for testing BCMA CAR T cells and tested for novel human BCMA CAR in efficacy studies. This study confirmed that several novel BCMA CARs (BCMA-4, BCMA-10, and BCMA-13) established an anti-tumor response in a xenograft model of multiple myeloma (Fig. 25A). Repeat the tumor experiment and test BCMA- 4. BCMA-10, BCMA-13 and BCMA-15. BCMA-4, BCMA-10, BCMA-13 and BCMA-15 demonstrated anti-tumor efficacy by inhibiting or reducing tumor growth at least 4 weeks (28 days) after implantation (Fig. 25B).

In addition, anti-tumor responses were associated with amplification of CD4 ⁺ CAR ⁺ and CD8 ⁺ CAR ⁺ T cells in peripheral blood of these mice (Figures 26A, B, C and D). For either of BCMA CAR, no anti-tumor efficacy was observed when this T cell expansion was not observed. BCMA-10, which exhibits the greatest anti-tumor efficacy in this model, also displays the most sustained CD8 ⁺ CAR ⁺ T cell expansion. The BCMA-4, BCMA-10, and BCMA-13 groups all showed significant changes in tumor growth compared to the control group. Lack of efficacy seen in the first tumor experiment with the tools BCMA CAR (BCMA-4NP) and BCMA-9 and BCMA-15 (Figure 25A) is associated with a lack of T cell expansion in peripheral blood in mice in these groups . Similarly, the lack of efficacy seen under BCMA-4NP in the second tumor experiment (Fig. 25B) was associated with a lack of T cell expansion in peripheral blood in mice.

The final bone marrow and spleen samples were also analyzed at the end of the in vivo tumor experiment to determine whether the CART pure line demonstrating anti-tumor efficacy exhibited differences in the ability to establish a bone marrow population compared to the group that did not exhibit anti-tumor efficacy. The number of CD4+ and CD8+ T cells expressing CAR was determined in the bone marrow (Figs. 27A and 27C) and spleen (Figs. 27B and 27D) of the mice from the first experiment. The results demonstrate that administration of BCMA-9 CART produces the highest number of CAR+ T cells (CD4+ and CD8+ T cells) in the bone marrow and spleen, indicating that BCMA-9 CART cells are efficiently expanded in vivo but have no killing or resistance Tumor activity. BCMA-10 CART cells display the most constant T cell establishment in the bone marrow and spleen. BCMA-4 and BCMA-15 CART cells were also found in the spleen.

Example 8: Identification of primary BCMA CAR constructs for therapy

To identify major BCMA CAR constructs, the results of several in vitro and in vivo analyses were analyzed. Experimental analysis, examples in which the details of the analysis are described, and the number of major BCMA CARs obtained after analyzing the analysis are summarized in the following table (Table 13). Analysis results are as shown The sequence analysis listed in 13 was performed to select candidate BCMA CARs that showed specificity, performance in immune effector cells, and activity in vitro and/or in vivo.

Based on in vitro analysis, eg, CL performance of Lentiviral transduction, JNL NFAT activation, T cell expansion, T cell proliferation, and target cell killing (as described in Examples 5, 6 and 7), 7 BCMA CAR identification For the primary CAR to be tested for efficacy in vivo, and in Example 8, 5 BCMA CARs (BCMA-4, BCMA-9, BCMA-10, BCMA-13, and BCMA-15) were tested. As described in Example 8, BCMA-4, BCMA-10, BCMA-13, and BCMA-15 all demonstrated antitumor efficacy, with BCMA-10 and BCMA-13 reproducibly demonstrating resistance in two separate in vivo experiments. Tumor efficacy.

Lentiviral force prices were compared between candidate BCMA CARs following automated virus generation and automated transduction of SupTl cells. Perform two independent lentiviral price analyses. The first force test procedure analyzed two independent DNA preparations (A and B) of BMCA-4, BCMA-10, BCMA-13 and BCMA-15 CAR. The second force test procedure analyzed three independent DNA preparations (A, B and C) of BMCA-4, BCMA-10, BCMA-13 and BCMA-15 CAR. Virus production is produced in an automated 96-well format. SupT1 fine Cell transduction was also performed via an automated 96-well format. CAR performance was manually analyzed by FACs and the results are shown in Figures 28A and 28B. All tested BCMA CARs showed similar levels and consistency of viral power.

Therefore, based on the results from in vitro and in vivo experiments as outlined in Table 13, BMCA-4, BCMA-10, BCMA-13, and BCMA-15 were identified as meeting the criteria for each analysis and it is highly anticipated that further testing will be used for treatment. use. When more stringent criteria were used for tumor regression analysis, the CAR constructs that reproducibly demonstrated anti-tumor efficacy in only two experiments were further analyzed, followed by identification of BCMA-10 and BCMA-13 for further therapeutic testing.

Example 9: Characterization of Major BCMA CAR Constructs

Additional analyses were performed to characterize the properties of the major BCMA CAR constructs BMCA-4, BCMA-10, BCMA-13, and BCMA-15 CAR that demonstrated in vitro and in vivo efficacy in the various assays described in Examples 5-8. Sequence alignment of BCMA-10 and BCMA-13 shows that both CARs have consensus heavy chain CDRs and a high degree of homology in the light chain CDRs. Competition analysis was performed between the four major candidates BMCA-4, BCMA-10, BCMA-13, and BCMA-15 CAR and BCMA-4NP (Tool CAR) as a control. BCMA-4NP was incubated with the BCMA substrate and combined between 50 and 300 seconds after incubation, as shown in FIG. Four BCMA CAR constructs were added and monitored for binding to the recipient. As shown in Figure 29, all four BCMA CAR constructs competed with the BCMA-4NP control, indicating that all four candidate BCMA CARs bind to the same epitope as the BCMA-4NP tool CAR. At a given concentration, if the candidate CAR binds to a different epitope, the RU change is expected to be about 70 RU. The small RU change observed during candidate BCMA CAR binding was attributed to a slight dissociation of the BCMA-4NP control sample from BCMA.

The antibody affinities of the following candidate BCMA CARs were also evaluated: BCMA-4, BCMA-10, BCMA-13 and BCMA-15. The results are shown in Figure 30 and are summarized in the table below.

BCMA-10 and BCMA-13 have similar affinities and are the lowest affinity for test candidates.

The selective binding of candidate BCMA CARs was also tested. BCMA is a receptor in the TNF receptor family, including closely related family members BaffR and TACI. BCMA has about 41% homology to BaffR and about 22% homology to TACI. T cells expressing candidate BCMA CAR BCMA-4, BCMA-10, BCMA-13, and BCMA-15 were incubated with recombinant BCMA, BaffR, or TACI fused to the Fc region. Binding was assessed by staining for CAR+ cells (Figure 31). The results showed that specific binding was observed only between all T cells expressing BCMA and recombinant BCMA-Fc, confirming that the BCMA CAR construct selectively binds to BCMA.

Example 10: BCMA performance in the brain

The tissue microarray results shown in Table 11 indicate that BCMA performance was detected in the cerebellum by immunohistochemical analysis. Human and non-human primate fumarin fixed paraffin-embedded (FFPE) brain tissue cultured with anti-BCMA antibodies against BCMA intracellular epitopes, such as USBio rabbit polyclonal antibody (0807-50G), And J6MO rabbit chimeric antibody staining that recognizes the extracellular epitope of BCMA. Staining with UsBio rabbit polyclonal antibody in brain tissue of non-human primates (cynomolgus macaques) caused positive staining of cerebellar crawling fibers (Fig. 32A) and cell bodies in the olive nucleus below the cerebellum (Fig. 32B). Staining non-human primate brain tissue with J6MO caused BCMA positive staining only in the lower olive nucleus (Fig. 32C; Ig control staining in Fig. 32E). Similarly, staining of human brain tissue with J6MO also caused positive staining of BCMA only in the lower olive nucleus (Fig. 32D; Ig control staining in Fig. 32F).

Immunohistochemistry results were confirmed by RNA analysis. In situ hybridization of non-human primates and human brain tissue. By in situ hybridization, both cerebellum and medulla were negative for BCMA by mRNA detection (Figures 33A and 33B). Quantitative PCR was also performed on non-human primates (cynomolgus macaques) and human cerebellum, medulla, stomach and kidney tissues. The qPCR results indicated that no BCMA mRNA was detected in the cerebellum and medulla of human (Fig. 33C) or non-human primate (Fig. 33D). The potential inconsistency between immunohistochemistry and RNA analysis can be attributed to different BCMA splice variants known in the art (Smirnova et al, Mol Immunol , 2008, 45: 1179-83).

RNAseq analysis was performed on normal tissues to detect all BCMA isoforms and splice variants. The results showed that BCMA was barely detected by RNAseq in normal tissues (Fig. 33E).

Further analysis was performed to determine if the protein detecting BCMA would be accessible to BCMA CART cells and to the effects of BCMA CART therapy. The PCR probe was redesigned and the performance of the BCMA splice variant was reassessed. Single cell RNAseq were performed in cerebellar samples. Confocal microscopy analysis was performed to observe intracellular staining. The effects of effective CART in mice, such as BCMA-10 and BCMA-13, on the brain were also assessed. To prevent the potential transport of BCMA CART cells to the brain, natalizumab can be administered to individuals.

Example 11: BCMA CART therapy in relapsed/refractory myeloma

This example provides a single, open-label pilot study to assess the safety and feasibility of autologous T cell infusions that exhibit BCMA-specific CAR in relapsed and/or refractory multiple myeloma. BCMA-CAR comprises a tandem TCRζ and a 4-1BB (TCRζ/4-1BB) co-stimulatory domain, and T cells expressing BCMA-CAR are referred to as BCMA CAR T cells.

Research objectives

The primary goal of this study was to determine the safety and tolerability of BCMA CAR T cells in MM patients. Secondary goals included: describing outcomes, including response rates, minimal residual disease (MRD) rates, progression-free and overall survival; and assessing the feasibility of manufacturing BCMA CAR T cells. Exploratory objectives include: about its expansion, persistence, homing, phenotype and merit Ability to characterize BCMA CAR T cells; evaluate the development of cellular and/or humoral immunity against BCMA CAR T cells; assess the effects of BCMA CAR T cells on B cells and plasma cell compartments, including immunoglobulin content; determine BCMA CAR Effect of T cells on systemic soluble immune factors in patients; assessment of BCMA performance on MM cells before and after treatment; and assessment of safety and efficacy of BCMA CAR T cell retreatment in patients who progressed after prior clinical benefit.

Study duration

The duration of active intervention and monitoring is approximately 2 years. After 2 years, according to FDA guidelines, monitoring of delayed adverse events will transform the long-term tracking program. The program will take approximately 12-18 months to complete the election.

Diagnosis and main inclusion criteria

Up to 12 evaluable individuals will be selected.

Inclusion criteria include at least 3 prior therapy routes that must include prior alkylating agents, proteasome inhibitors (PIs), and immunomodulatory drugs (IMiD) (or IMID (immunomodulatory drugs, thalidomide, and lysine) Adults with 18 years of age with relapsed and/or refractory multiple myeloma after two refractory treatments with prodrugs and proteasome inhibitors. After the most recent protocol, the patient relapsed, defined as the IMWG (International Myeloma Working Group) standard for PD, or refractory, defined as achieving <PR). Patients have a limited prognosis under currently available therapies ( 2 years expected to survive).

Research products, dosages, routes, protocols

A single infusion of BCMA CAR T cells was administered by intravenous infusion. Group 1 will receive 1-5 x 10 ⁸ BCMA CAR T cells alone, calculated as the range of 2%-50% transduced cells in total cells (if there is unexpected severe toxicity, the cell dose in group 1 can be reduced to 1-5×10 ⁷ BCMA CAR T cells (group-1). Group 2 will receive cyclophosphamide (Site sylvestre) 1.5 1-3 days before infusion of 1-5×10 ⁷ BCMA CAR T cells. g/m ² , administered by intravenous infusion. Group 1-3 will receive cyclophosphamide 1.5g/m ² 1-3 days before infusion of 1-5×10 ⁸ BCMA CAR T cells, by intravenous Infusion of investment.

Based on the total volume to be infused and the recommended infusion rate of 10-20 mL per minute. The dosages and schedules for each group are summarized in the table below and the schematic is shown in Figure 34.

To prevent the potential transport of T cells to the brain, natalizumab (TYSABRI®) can be administered to patients.

Patient monitoring

Tumor response was measured by serum and urine protein electrophoresis and immunofixation, bone marrow biopsy, and imaging (if skeletal lesions were present prior to treatment). A neurological examination will also be performed before and after the therapy to ensure no neurological changes.

Statistical methods

Statistical analysis will be primarily descriptive and consistent with the nature of the research. Descriptive statistics will be used to determine the relative portability, persistence, and transport of the study drug components to the blood and bone marrow. All adverse events will be described and an accurate 95% confidence interval (for all and major categories) will be generated for adverse event rates. Analysis of other secondary endpoints such as anti-tumor activity will also be primarily descriptive and may include summary statistics such as mean and standard deviation for survival information or Kaplan-Meier curve.

Example 12: Secretion of cytokines from CART cells

The ability of CART cells to respond to target secretion of cytokines is determined. CART cells containing BCMA-10 CAR or untransduced T cells were co-cultured with BCMA-positive (KMS11-luc) or BCMA-negative (U87-luc) target cells, and IL-2, IFNγ and TNFα were measured to the medium. Secretion. Specifically, thawing CAR T cells containing BCMA-10 CAR or untransduced were co-cultured with target cells for 20 hours at an effector:target ratio of 2.5:1. Target cells include BCMA positive luciferylated KMS-11 (KMS11-luc) or BCMA positive luciferylated U87 cells (U87-luc). Culture plates with effector cells 3 × 10 ⁴ th target T cells in complete cell culture medium in a total volume of 200 [mu] l / well of a 96-well U-bottom. After 20 hours, the supernatant was removed from the culture and IFNγ, IL-2 and TNFα secretion was quantified on a FACS by cytology bead array (BD Bioscience) according to the manufacturer's instructions. Measured in duplicate. Error bars indicate standard deviation (Figures 35A-35B). The results demonstrate that BCMA-10 CART, but not transduced T cells, are stimulated to produce cytokines by expressing BCMA, but not BCMA-negative target cells (Figures 35A-35B).

Example 13: Function of BCMA-CART in multiple myeloma

Multiple myeloma (MM) is a malignant disease of plasma cells in the bone marrow, including clinical features of anemia, skin lesions, skeletal tenderness or pain, fatigue, osteolytic lesions, hypercalcemia, renal failure, and recurrent bacterial infections. Most common. Although recent treatment with drugs such as lenalidomide has resulted in a significant increase in the survival of recurrent MM, the disease is almost always incurable. The median 5-year survival rate was approximately 35%. Due to poor prognosis, an effective targeted therapy is needed.

Treatment with T cells engineered to express chimeric antigen receptor (CAR) can produce promising immunotherapies for hematological malignancies such as ALL. CAR contains a fusion protein that recognizes a cell surface target protein expressed on a tumor cell. Differential gene expression studies have identified B cell mature antigens (BCMA, CD269) as highly specific target antigens for malignant plasma cells and normal plasma cells; therefore, BCMA is a potential target antigen for CAR T cell therapy. See, for example, Carpenter et al. Clin Cancer Res. 19.8 (2013): 2048-60.

BCMA is a member of the TNF receptor superfamily. The protein is encoded by the TNFRSF17 gene. BCMA is expressed in mature B lymphocytes. It binds to tumor necrosis factor (ligand) superfamily member 13b (TNFSF13B/TALL-1/BAFF), APRIL and various TRAF family members. Interaction with its ligand produces NF-κB and MAPK8/JNK signals, which are involved in B cell development, long-term plasma survival, and cell proliferation.

This example describes a preclinical study evaluating the in vitro and in vivo function of huBCMA-BBz CAR transduced T cells (CART-BCMA or BCMA-CART) incorporating BCMA10 scFv.

material

T cells. T cells from healthy donors were obtained from the University of Pennsylvania CFAR Human Immunology Core (Philadelphia, PA). Cells were prepared by leukocyte depletion of healthy volunteer donors.

Medium. RPMI medium (Gibco) supplemented with 10% fetal bovine serum and filtered (Valley Biomedical), 2 mM GlutaMax (Invitrogen), 10 mM HEPES (Invitrogen), 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco) was used.

CD3/28 beads. CD3/28 beads (GMP grade) were made by the Clinical Cell and Vaccine Production Facility of the University of Pennsylvania.

Plastid. The huBCMA-BBz CAR construct cloned in the pELPS lentiviral vector NVP-MCM998 was generated by Novartis.

Antibody. Antibody goat anti-human BCMA PE label (Biolegend Cat. No. 357504) and streptavidin (BD Biosciences) were used to detect BCMA expression on multiple myeloma cell lines. For CAR T cell expression, BCMA fc fusion protein (R&D Systems, Cat. No. 193-BC-050) was used followed by anti-human IgG fc-PE antibody (Biolengend, Cat. No. 409304).

PBS. PBS from Gibco.

Lentiviral packaging. huBCMA-BBz was slowed by transfection with lipofectamine 2000 (Invitrogen catalog number 11668027) on 293T cells using PCL USUG, PRSV Rev, PGAG pol plastid (Nature Technology corp. catalog number NTC RP20) Virus preparation.

Cell line. Human embryonic kidney 293T cells (ATCC catalog number CRL-3216) were used for lentiviral preparation. Multiple myeloma cell line RPMI 8226 (ATCC catalog number CCL-155), MM1S (ATCC catalog number CRL-2974), U266 (ATCC catalog number CRL-3216), NCI H929 (ATCC catalog number CRL-9068), and OPM2 ( DSMZ catalog number ACC-50) and K562 (ATCC catalog number CCL-256) or K562-BCMA cells (BCMA lentiviral vector from Genecopoeia, catalog number Lv105) were used for functional experiments on CART-BCMA cells.

method

Lentiviral production protocol and potency assay. 293T cells were cultured in RPMI 1640 medium supplemented with 10% FBS (ATCC Cat. No. 30-2020) and Streptococcus/penicillin (Invitrogen Cat. No. 10378-016) (Gibco Cat. No. 11875-080) T150 flask (Corning Costar Cat. No. 430825) was inoculated with 8×10 ⁶ cells per flask, and transfected with huBCMA-BBz encoding pELPS lentiviral vector for 24 hours via encapsulated plastid mixture (Nature Technology corp.). . The resulting virus preparation was stored at -80 °C. The recombinant lentivirus was titrated on CD4T.

Transduction protocol. T cells obtained from Human Immunology Core were washed once in culture medium, resuspended at 10 ⁶ cells/ml, and stimulated with CD3/28 beads at a 1:3 cell:bead ratio. On day 2, lentiviral transduction was performed by mixing the lentiviral vector into cell culture with MOI 3.

T cell transduction. Stimulates T cells to feed, and divides into 0.8 × 10 ⁶ cells / ml every 2-3 days, for 7-9 days, or until the cells are still, such as by cell division rate reduction and average The cell volume was reduced to less than about 300 fl.

Cell cultures. Multiple myeloma cell lines and K562, K562 BCMA cell lines were cultured in RPMI with 10% FBS and antibiotics.

Cell count. During amplification, cells were counted by gently mixing the culture and 40 μl of cells were collected from the culture volume, and cells were counted every 3 days and placed in an accuvette (Beckman Coulter) with 20 ml of Isoton II dilution buffer for use. Coulter Multisizer 3 (Beckman Coulter) counts. The results of this test (absolute cell number and cell volume) were used to determine cell concentration, total cell number, growth rate, and dilution volume.

⁵¹ Cr release assay. The ability of CART-BCMA cells to kill target cells expressing BCMA was assessed using a ⁵¹ Cr release assay. Briefly, target K562-BCMA cells (or control K562 cells) and multiple myeloma cell lines were labeled with ⁵¹ Cr (sodium dichromate) washed with effector CART-BCMA or control untransduced T Cells (NTD) were co-cultured at different effect/target ratios. Supernatants were collected at 4 hours and placed in 96-well Lumaplate (Perkin Elmer). The amount of ⁵¹ Cr released from the labeled target cells was measured on a liquid scintillation counter (MicroBeta trilux, Perkin Elmer). Target cells incubated in medium alone or with 1% SDS were used to determine spontaneous (S) or maximum (M) ⁵¹ Ci release. The specific percentage of dissolution was calculated as follows: 100 x (cpm experimental release - cpm S release) / (cpm M release - cpm S release).

CAR detection on transduced T cells. To assess transduction of CART-BCMA, T cells were stained with BCMA-Fc fusion protein (R&D Systems) followed by anti-human IgG Fc-PE antibody (Biolengend).

Flow cytometry. For anti-BCMA staining, human myeloma cell lines were stained with goat anti-human-PE BCMA antibody (Bioloegend) followed by streptavidin (BD Biosciences). Flow cytometry analysis of all experiments was performed by using FlowJo (Tree Star, Inc.).

ELISA. Target K562-BCMA cells (or control K562 cells) or multiple myeloma cell lines and CAR-transduced T cells in a double well of a 96-well flat bottom plate at a 1:3 target: effect ratio . ELISA analysis was performed by collecting a 1:10 dilution of the supernatant after 16 hours of incubation using a human IFNy or IL2 Duoset ELISA kit (R&D) as recommended by the manufacturer.

result

huBCMA-BBz is highly expressed on transduced T cells.

Freshly purified negatively selected normal human T cells were activated and expanded in vitro using CD3/28 beads (1:3 cells: bead ratio). On day 1 post-activation, cells were transduced or mock-transduced with a pre-clinical lentiviral vector expressing huBMCA-BBz (NTD control). Figure 36A shows the increase in total T cells during culture. BCMA-CART cells were counted every 3 days and the ratio of dividing cells was adjusted. T cells were counted using a Coulter counter Multisizer III and fed every 2 days until the end of the amplification cycle (days 7-9). On day 6 of ex vivo expansion, 200 μl of CART-BCMA or control NTD T cells were stained as described in the Methods section above. Use FCS and SSC to gate live cell populations. Flow acquisition was performed on a BC FACS Canto instrument and flow analysis was performed using FlowJo software (TreeStar, Inc).

No difference in proliferation rates of untransduced (NTD) and CART-BCMA cells was observed, indicating that lentiviral performance did not affect the proliferative potential of T cells (Fig. 36A). Transduction efficiency was assessed on day 6 after transduction as described in the Methods section above. Figure 36B shows that the huBCMA-BBz transduction efficiency is 49%. These results confirm that huBCMA-BBz CAR is effective on the surface of human T cells.

BCMA has different expression levels on multiple myeloma cell lines.

The BCMA surface appearance on multiple myeloma cell lines was determined by flow cytometry staining. The live cell population is gated by FCS/SCC parameters. Flow acquisition was performed on a canto instrument and flow analysis was performed using FlowJo software. Most of the multiple myeloma tested showed strong BCMA performance, while the surface BCMA performance of RPMI 8226 and control K562-BCMA cells was lower (Figure 37). For all graphs, the orange solid peaks indicate isotype controls and the blue solid peaks were stained with BCMA antibody (Figure 37). Flow cytometry staining revealed BCMA expression on the surface of multiple myeloma cell lines NCI H929, U266, RPMI 8226, OPM2 and MM1S as well as K562-BCMA cells. No BCMA was detected on the surface of the K562 cell line (Fig. 37). These results demonstrate that BCMA is represented by several multiple myeloma cell lines with a change in performance of about 1 log.

CART-BCMA cells specifically respond to different multiple myeloma cell lines expressing BCMA to produce cytokines and exhibit cytotoxic properties.

The ability of CART-BCMA cells to produce cytokines and kill BCMA+ target cells was determined. CART-BCMA cells (huBCMA-BBz transduced T cells) or control untransduced T cells (NTD) in duplicate with K562, K562-BCMA or multiple myeloma cell lines (MM1S, OPM2 or U266) Incubate for 16 hours. Cells were co-cultured at a 3:1 T cell:target cell ratio. The cell-free supernatant was harvested and the production of IL2 or IFNy was assessed as described in the Methods section above.

In the presence of K562 (K562-BCMA) engineered to express BCMA, CART-BCMA cells specifically produce IL2 or IFNy compared to wild-type K562 cells that do not exhibit antigen. CART-BCMA cells were also able to produce cytokines in the presence of U266, OPM2 and MM1S multiple myeloma cell lines (Fig. 38A-38B). These results demonstrate that CART-BCMA cells produce pro-inflammatory cytokines in the presence of BCMA+ target cell lines.

Another in vitro measure of the anti-tumor efficacy of CART-BCMA cells is its ability to kill BCMA+ target cells. The T cells are activated and transduced as described above, for example, with respect to Figures 36A-36B. BCMA10 CAR T cells and ⁵¹ Cr-labeled K562-BCMA, RPMI 8226 or MM1S cell lines are shown in Figure 39A-39C as effector cells: target ratio (E:T ratio) (0, 10, 20 or 30 E) :T) Co-culture for 4 hours and calculate the percentage of dissolution as described in the Methods section above. Figure 39A shows that CART-BCMA cells specifically kill K562-BCMA cells. Analysis of the CART-BCMA cytotoxic cells at 4 hours is also effective to kill BCMA ^high MM1S multiple myeloma cell line RPMI 8226 and BCMA ^low cell lines (FIG. 39B, 39C). These results confirmed that CART-BCMA showed enhanced cytotoxic activity against multiple myeloma cell lines ( ⁵¹ Cr assay).

Antitumor activity of CART-BCMA cells in vivo

Using a BCMI 8226 cell that exhibits lower BCMA performance compared to many other myeloma cell lines, a mouse model was established to assess CART-BCMA recognition and elimination by engineering to express deductive green luciferase ( CBG) The ability of tumors produced by BCMA ^low RPMI 8226 cell line for bioluminescence imaging (BLI). On day 30 after tumor cell inoculation, NOD/SCID/γ-chain ^-/- (NSG) mice with established intravenous RPMI 8226 tumors received intravenous CART-BCMA cell injection (N=10) or untransduced controls. T cells (NTD) (N=10). NSG mice transplanted with RPMI-8226 cells were treated with 5 x 10 ⁶ CART-BCMA T cells on day 30 after injection of tumor cells. At 9 weeks after infusion of T cells (week 14 after tumor injection), myeloma progression was followed by BLI in vivo (BLI in the abdominal and dorsal mouse areas once a week).

Untransduced T cells failed to control the tumor, and all mice must be sacrificed due to disease progression at weeks 10-11 after tumor injection (Figures 40A and 40C). In contrast, mice receiving CART-BCMA cells exhibited control of tumor growth in most mice compared to 0% survival of NTD control treated mice, and CART at 10 weeks after tumor cell inoculation. - BCMA treated mice survived 80% (Figures 40B and 40C). These results confirmed that intramedullary RPMI 8226 tumors were inhibited by CART-BCMA treatment.

Example 14: BCMA-CART Dosing Protocol

BCMA CART cell therapy, such as the BCMA CART cell therapy described herein, can be administered to a patient, such as a multiple myeloma patient, according to the dosing regimen described herein, such as the dosing regimen described below.

Leukocyte depletion was performed on the patient prior to receiving BCMA CART therapy to obtain autologous T cells. Manufacture of BCMA lentiCAR T cells and/or cryopreservation. The patient can receive therapy during manufacturing to maintain disease control. Some patients may receive lymphocyte depletion therapy (eg, Settle&apos;s) prior to administration of CART cells. For example, lymphocyte depletion chemotherapy, e.g. Dorset fir (e.g., 1.5g / m ²⁾ administered to the patient. In other dosing regimens, lymphocyte depletion chemotherapy is not administered to the patient. The patient is then treated with BCMA CART cells according to the dosing regimen described herein.

The dosing regimen involves dose partitioning, such as where a certain percentage of the total cell dose is delivered on the first day of treatment, a different percentage of the total cell dose is delivered on the subsequent treatment day, and a different percentage of the total cell dose is delivered on a subsequent therapeutic day. For example, 10% of the total cell dose is delivered on the first day, 30% of the total cell dose is delivered on the second day, and the remaining 60% of the total cell dose is delivered on the third day of treatment. For example, the total cell dose includes 1 to 5 x 10 ⁷ or 1 to 5 x 10 ⁸ BCMA-CART cells.

In one embodiment the administration is not administered lymphocyte depletion chemotherapy, and administration of a BCMA-CART to a total dose of 5 × 10 ⁷ cells of th (e.g. by infusion), wherein the first day of treatment administered to cells 10% of the dose, 30% administered on the second day of treatment, and 60% on the third day of treatment. In another dosing regimen, not administered lymphocyte depletion chemotherapy, and administration of a BCMA-CART to a total dose of cells (e.g. by infusion), wherein the first day of treatment and the administration of 5 × 10 ⁸ cells 10% of the dose, 30% administered on the second day of treatment, and 60% on the third day of treatment. In another dosing regimen, lymphocyte depletion therapy (Settling, at 1.5 g/m ² ) was administered three days prior to administration of BCMA-CART cells, and then 1 to 5 x 10 ⁷ were administered. The total BCMA-CART cell dose (e.g., by infusion), wherein 10% of the cell dose is administered on the first day of treatment, 30% is administered on the second day of treatment, and 60% is administered on the third day of treatment. In another dosing regimen, lymphocyte depletion therapy (Settling, at 1.5 g/m ² ) was administered three days prior to administration of BCMA-CART cells, and then 1 to 5 x 10 ⁸ were administered. The total BCMA-CART cell dose (e.g., by infusion), wherein 10% of the cell dose is administered on the first day of treatment, 30% is administered on the second day of treatment, and 60% is administered on the third day of treatment.

Clinical laboratory evaluations were performed on days 1, 2, 4, 7, 14, 21, 28, every 4 weeks after administration of CART cells (where day 0 was the first day of CART administration). Multiple myeloma assessment was performed prior to CART administration, on the first day (day 0) and on days 14, 28 and every 4 weeks of CART administration. Bone marrow aspiration/biopsy (bx) was performed before CART administration and on days 28 and 90 after CART administration. After 28 days of CART treatment, follow-up every 4 weeks for up to 6 months, followed by tracking every 3 months for up to 2 years.

Example 15: Low dose RAD001 stimulates CART proliferation in a cell culture model

The effect of low dose RAD001 on proliferation of CAR T cells in vitro was evaluated by co-culturing cells expressing CART with target cells in the presence of different concentrations of RAD001.

Materials and Methods Generation of CAR-transduced T cells

The humanized anti-human CD19 CAR (huCART19) lentiviral transfer vector was used to generate genomic material encapsulated into VSVg pseudo lentiviral particles. The amino acid and nucleotide sequence of the humanized anti-human CD19 CAR (huCART19) is CAR 1, ID 104875, as described in PCT Publication No. WO 2014/153270, filed on Mar. NO.85 and 31.

The lentiviral transfer vector DNA was mixed with the three encapsulating components VSVg env, gag/pol and rev, VSVg env, gag/pol and rev were combined with the lipoamine reagent to transfect Lenti-X 293T cells. The medium was changed after 24 hours and 30 hours thereafter, the virus-containing medium was collected, filtered and stored at -80 °C. CART is produced by transduction of fresh or frozen raw T cells obtained by healthy donor blood or negative magnetic selection of leukopak. T cells were activated by incubation with anti-CD3/anti-CD28 beads for 24 hours, followed by addition of virus supernatant or concentrated virus (MOI = 2 or 10, respectively) to the culture. The modified T cells were expanded for about 10 days. The percentage of transduced cells (expressing CAR on the cell surface) and the amount of CAR expression (relative fluorescence intensity, geometric mean) were determined by flow cytology analysis between day 7 and day 9. The combination of slower growth rate and T cell size approaching approximately 350 fL determines the status of T cells to be cryopreserved for subsequent analysis.

Assess the proliferation of CART

To assess the functionality of CART, T cells were thawed and counted, and viability was assessed by Cellometer. The number of CAR positive cells in each culture was corrected using untransduced T cells (UTD). Start with 50 nM and titrate with RAD001 to test the effect of RAD001 on CART. The target cell line used in all co-culture experiments was Nalm-6, a human pre-B cell acute lymphoblastian that expresses CD19 and is transduced to express luciferase. Cellular leukemia (ALL) cell line.

To measure proliferation of CART, T cells were incubated with target cells at a ratio of 1:1. The assay was run for 4 days at which time the cells were stained for CD3, CD4, CD8 and CAR performance. The number of T cells was assessed by flow cytometry using counting beads as a reference.

result

The proliferative capacity of CART cells was tested in a 4-day co-culture assay. The number of CAR-positive CD3-positive T cells (dark bars) and total CD3-positive T cells (shallow bars) was assessed after CAR transduction and untransduced T cells were cultured with Nalm-6 (Fig. 43). huCART19 cells were expanded when cultured in the presence of less than 0.016 nM RAD001, and to a lesser extent at higher concentrations of the compound. Importantly, the proliferation at 0.0032 and 0.016 nM RAD001 was higher than that observed without the addition of RAD001. Untransduced T cells (UTD) did not display detectable amplification.

Example 16: Low dose RAD001 stimulated CART amplification in vivo

This example assesses the ability of huCAR19 cells to proliferate in vivo at different concentrations of RAD001.

Materials and Methods:

NALM6-luc cells: NALM6 human acute lymphoblastic leukemia (ALL) cell line was produced from peripheral blood of patients with relapsed ALL. The cells were then labeled with firefly luciferase. These suspension cells were grown in RPMI supplemented with 10% heat-inactivated fetal bovine serum.

Mice: 6 weeks old ^{NSG (NOD.Cg- Prkdc scid Il2rg tm1Wjl /} SzJ) mice were received from Jackson Laboratory (stock number 005557).

Tumor Implantation: NALM6-luc cells were grown and expanded in vitro in RPMI supplemented with 10% heat-inactivated fetal bovine serum. The cells were then transferred to a 15 ml conical tube and washed twice with cold sterile PBS. Next, NALM6-luc cells were counted and at a concentration of 10 × 10 ⁶ cells per ml of PBS and resuspended. The cells were placed on ice and immediately (into one hour) implanted into the mice. NALM6-luc cells were injected intravenously via the tail vein in a volume of 100 μl, for a total of 1 × 10 ⁶ cells per mouse.

CAR T cells were administered: 5 x 10 ⁶ CAR T cells were administered to mice 7 days after tumor implantation. The cells were decomposed in the middle of a 37 ° C water bath and then completely thawed by adding 1 ml of cold sterile PBS to the tubes containing the cells. The thawed cells were transferred to a 15 ml falcon tube and adjusted to a final volume of 10 ml with PBS. The cells were washed twice at 1000 rpm for 10 minutes each, followed by counting on a hemocytometer. T cells were then a concentration of 50 × 10 ⁶ cells per ml of cold PBS and resuspended and kept on ice until administered mice. Mice were intravenously injected with 100 μl of CAR T cells via the tail vein at a dose of 5 × 10 ⁶ CAR T cells per mouse. Eight mice of each group were treated with 100 μl of PBS alone (PBS) or humanized CD19 CAR T cells.

RAD001 given: 1mg equal RAD001 formulation of 50mg and then concentrated microemulsions when administered resuspended in D5W (5% dextrose in water of) the. Mice were orally administered (via oral gavage) daily to the required dose of 200 μl RAD001.

PK analysis: On day 7 after tumor implantation, mice were given daily RAD001. The administration groups were as follows: 0.3 mg/kg, 1 mg/kg, 3 mg/kg, and 10 mg/kg. On days 0 and 14, the following time points after the first dose and the last dose of RAD001, the mice were bled for PK analysis: 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours and 24 hours.

result:

Amplification and pharmacokinetics of RAD001 were tested in NSG mice with NALM6-luc tumors. Daily oral administration of RAD001 alone did not affect the growth of NALM6-luc tumors (Fig. 44). Pharmacokinetic analysis of RAD001 showed that it was quite stable in the blood of tumor-loaded mice (Figures 45A and 45B). PK analysis on day 0 and day 14 showed that the concentration of RAD001 in the blood exceeded 10 nm, even at the lowest test dose (0.3 mg/kg). After hours.

Based on these doses, huCAR19 CAR T cells were administered with and without RAD001 to determine the proliferative capacity of these cells. The highest dose used was 3 mg/kg based on the amount of RAD001 in the blood after 24 hours of administration. Because the RAD001 concentration exceeded 10 nM after 24 hours of the last dose of RAD001, several lower doses of RAD001 were used for in vivo studies of CAR T cells. At the beginning of the daily oral administration of RAD001, IV was given to the CAR T cells. Mouse T cell expansion was monitored via FACS.

The lowest dose of RAD001 demonstrated enhanced proliferation of CAR T cells (Figure 46). This enhanced proliferation is more pronounced and longer under CD4 ⁺ CAR T cells than CD8 ⁺ CAR T cells. However, in the CD8 ⁺ CAR T cells, the enhanced visibility early time point after proliferation CAR T cell dose.

Example 17: CD19 CAR T cells for the treatment of multiple myeloma

Even with the current regimen of chemotherapy, targeted therapy, and autologous stem cell transplantation, myeloma is considered an incurable disease. This example describes the treatment of multiple myeloma (MM) with autologous T cells directed against CD19 and a chimeric antigen receptor (lentivirus/CD19: 4-1BB: CD3ζ; also known as "CART19" or CTL019). This example demonstrates that based on myeloma stem cells and/or tumor cells that target very low levels of CD19 (undetectable by most methods), CAR therapy for CD19 can establish deep and long-lasting relief.

In the treatment of patients with invasive secondary plasma cell leukemia, it was found that CART19 administered two days after remediation of autologous stem cell transplantation caused rapid clearance of plasma cell leukemia in patients who had progressed through multiline chemotherapy, and that The reaction is excellent. Before treatment, the patient relied on blood transfusion for several months; in the two months after treatment, he had recovered his blood count (with normal range of platelet counts and white blood cell counts) and had no blood transfusion because he was discharged from hospital and was relieved of treatment. .

Since myeloma cells do not naturally exhibit CD19, the discovery that CART19 treatment induces a rapid and significant tumor response in this tumor is unexpected. Do not wish to be subject to a particular theory Binding, inferred that CART19 can be used to treat myeloma because: (1) Although myeloma cells are traditionally considered to be negative for CD19 by flow cytometry, there is evidence that myeloma cells can exhibit very low levels of CD19, Such that the expression is detectable by RNA but not detectable by flow cytometry or immunohistochemistry; and (2) the concept of targeting pure lineage B cells, which is considered to cause multiple myeloma Cancer stem cells, and are particularly resistant to chemotherapy. Although there is a pure relationship between B cells and myeloma tumor cells, traditional myeloma therapy targets malignant plasma cells rather than B cells. Thus, CART19 for the treatment of myeloma targets a different population of cells than most myeloma therapies.

In a single patient experience, the patient has circulating plasma cells and is able to test the CD19 performance of his tumor cells. About 1-2% of its tumor cells express CD19 antigen (Fig. 47). Therefore, it is inferred that CART19 has a direct effect on a very small population of its tumor cells; some reactions are excellent, but based on a very small population targeting only CD19+ tumor cells, it has not been predicted.

In this case, CART19 was administered after autologous stem cell transplantation first aid after high dose melphalan. Although this is the standard treatment in myeloma, it is not curative. In addition, this patient has previously been transplanted with tandem autologous stem cells and relapsed early (<6 months) after transplantation. Without wishing to be bound by a particular theory, the use of CART19 cells as described in this example may have a non-overlapping mechanism in the treatment of myeloma when combined with remedial autologous stem cell transplantation.

Patients with refractory multiple myeloma were treated with CTL019 after myeloablative chemotherapy and ASCT. Despite the detectable loss of CTL019 and normal CD19-positive B-cell recovery, maintenance was relieved, indicating that this response does not require sustained CTL019 activity. In addition, even though both flow cytometry and RT-PCR, most (99.95%) of the neoplastic plasma cells were negative for CD19, and this patient's response was also achieved.

The lack of detectable CD19 expression in this patient's dominant neoplastic plasma cell population indicates that the clinically relevant CTL 019 target is outside this dominant CD19 negative population. Multiple bone The neoplastic plasma cells in myeloma patients show genetic, immunophenotypic and functional heterogeneity. The survival of a pure line through anti-myeloma therapy may require a specific subpopulation. Among the patients reported herein, for example, a small subset of plasma cells expressing CD19 may have relative melphalan resistance but are sensitive to CTL019. This finding suggests that a small subset of therapeutically targeted pure lines can produce long-lasting clinical benefits when combined with conventional anti-myeloma therapies.

Alternatively, the clinically relevant target of CTL019 in this patient may be located outside of the population of neoplastic plasma cells. For example, CTL019 can target a population of stem cells that are relatively small but produce neoplastic plasma cells. Therefore, multiple myeloma can be a plurality of advanced B lineage cell types, not just diseases of terminally differentiated plasma cells, so that the therapy of CTL019 such as B lymphocytes can be applied directly to the therapy of plasma cells. Adjuvant.

In the Phase I trial, CART19 was used to treat an additional ten patients with multiple myeloma, and at least three patients have been treated to date.

Basic principle of dose

In the first three patients at a dose in the range of cells of the CART-19 1.4 × 10 ⁷ to 1.1 × 10 ⁹ has been observed in clinical activity. This observation confirmed that there was no significant dose-response relationship in at least the first three patients treated. A complete response was observed in patients who received two log multiples of the dose difference. Thus, unlike standard drugs that are metabolized, CAR T cells can have a wide dose response range. This is most likely because CAR T cells can proliferate extensively in patients. A dose range of 1-5 x 10 ⁸ CART-19 cells was therefore set for infusion. In this single patient study based on Enci therapy, up to 5 x 10 ⁸ CART19 cells were provided with no dose lower limit. For tests of ten patients, will provide patients 1-5 × 10 ⁷ th CART-19 cells.

General design

This is a single patient study based on Enci therapy; a model was established after the Phase I study to determine if the transduced can be safely infused to express CART-19 autologous T cells. The primary goal of the study was to remedy the early relapse after the first ASCT The safety, tolerability and transplant potential of CART-19T cells were determined in ASCT patients. The program consists of open-label lead research.

Initially, the individual will undergo a bone marrow biopsy and perform routine laboratory and imaging evaluations of their MM. Eligible individuals will undergo steady-state clearance to obtain a large number of peripheral blood mononuclear cells (PBMC) to make CART-19. T cells were purified from PBMCs, transduced with TCR(R)/4-1BB lentiviral vector, expanded in vitro and then frozen for future administration. The results of comparing the number of patients with T cells collected, expanded, or under-produced to the number of patients who have successfully produced T cells are recorded; product manufacturing feasibility issues are not expected in this patient population.

Individuals have generally maintained sufficient peripheral blood stem cells for movement/collection from their first ASCT preparation to perform two additional ASCTs. The second movement/collection procedure is not performed before or after its steady-state clearance according to the preferred protocol of the treating physician. About two weeks after the initial leukocyte depletion, the individual will enter the hospital and receive a high dose of melphalan (-2 days), followed by two days (day 0) infusion of autologous stem cells, and all individuals will be between twelve and ten Four days later (day +12-14), an infusion of CART-19 cells was received. Up to 10 patients will be enrolled.

All individuals will have a blood test to assess the safety and portability and persistence of CART-19 cells at regular intervals through the 4th week of the study. On days +42 and +100, individuals will undergo a bone marrow aspiration/biopsy to assess bone marrow plasma cell load and transport of CART-19 cells to bone marrow. On day 100, a formal response assessment was performed according to International Myeloma Working Group (IMWG) Standard 136, and TTP was monitored for patients with multiple myeloma according to routine clinical procedures. The primary efficacy outcome measured in this study will be the TTP after the patient's initial ASCT and the TTP after ASCT in this study.

Treatment programs Recurrent/progressive multiple myeloma therapy

Patients can receive relapsed/progressive multiple myeloma therapy prior to enrollment based on their preferences. The therapy can continue at the time of the election.

Patients must stop all treatments two weeks before the procedure and two weeks before the high dose of melphalan. If the time between the clearance and the high dose of melphalan is expected to be more than two weeks, the patient may be treated at his discretion by the treating physician to resume the therapy after the removal.

High dose melphalan (day -2)

On day -3 or -2, the patient will enter the hospital and will be examined by the attending physician and routine laboratory tests prior to starting the treatment protocol, which will include monitoring the parameters of the tumor lysis syndrome. If such tests did not draw blood within 7 days of entry, blood was drawn for MM monitoring laboratory testing (SPEP, quantitative immunoglobulin, and serum free light chain analysis) prior to initiation of therapy.

High dose therapy will consist of intravenous administration of a dose of 200 mg/m ² of melphalan on day -2 for more than about 20 minutes. The melphalan dose will be reduced to 140 mg/m ² for patients >70 years of age or for patients of any age who are not able to tolerate a dose of 200 mg/m ² at the discretion of the treating physician. All patients will receive standard anti-vomiting prophylactic agents, which may include dexamethasone and standard antibiotic prophylactic agents.

Stem cell reinfusion (Day 0)

On day 0, stem cell infusion will be performed at least 18 hours after administration of high dose melphalan. Stem cells will be infused intravenously for more than about 20-60 minutes after pre-operative administration according to standard protocol procedures. Infusion per kg body weight should be at least 2 × 10 ⁶ th CD34 + progenitor cells. Further, when the advanced or delayed graft transplant failure, per kg body weight is at least 1 × 10 ⁶ th CD34 + progenitor cells should be available as a supporting stem cell product. Starting on day +5, the G-CSF should be administered to SQ and given according to standard institutional operations. Other supportive care measures, such as infusion support, will be conducted in accordance with standard agency guidelines.

CART19 cell infusion (day +12-14)

With a single dose will CART-19 transduced via the T cell, consisting of up to 5 × 10 ⁷ th CART-19 cells. Acceptable minimum by infusion of the vector-transduced CD19 TCRζ4-1BB cell dose of 1 × 10 ⁷ th. CART-19 cells will be administered in a single dose by rapid intravenous infusion on days +12-14 after stem cell infusion. If the patient does not meet any of the inclusion criteria described herein at the 12-14 day window, the CART-19 infusion may be delayed beyond the +12-14 days until the criteria are met.

Maintain lenalidomide

Individuals who receive and tolerate maintenance of lenalidomide after the first ASCT will initiate lenalidomide maintenance therapy on approximately +100 days, assuming that the treating physician has no contraindications. The initial dose will be 10 mg per day unless previously experienced to specify an alternate starting dose for a particular patient. Maintenance therapy will continue until disease progression or intolerance.

Investing in research drugs

The infusion will be performed in a separate room in Rhoads using protective measures from immunosuppressed patients. Transduced T cells will be administered by rapid intravenous infusion via a 18 gauge latex-free Y-type blood device with a 3-way stopcock at a flow rate of about 10 mL to 20 ml per minute. The duration of the infusion will be based on the total volume to be infused and the recommended infusion rate. Each infusion bag will adhere to the following label: "For self-use only". In addition, the tag will have at least two unique identifiers, such as an abbreviation of the individual, date of birth, and study number. Before the infusion, the two individuals will independently test all of this information in the presence of the individual and thus confirm that the information correctly matches the participant.

Package

Containing a single dose infusion of 1-5 × 10 ⁷ of CA T19 transduced cells, wherein smallest 1 × 10 ⁷ cells of the CART-19 dose acceptable for infusion. Each bag will contain an aliquot of cryo medium (volume dependent dose) containing the following infusible grade reagent (% v/v): 31.25% plasma-te-A, 31.25% right-handed Sugar (5%), 0.45% NaCl, up to 7.5% DMSO, 1% polydextrose 40, 5% human serum albumin.

Clearance

Perform a large volume (12-15 liters or 4-6 blood volume) removal procedure at the removal center. PBMC were obtained for CART-19 during this procedure. It is intended to produce at least 5 x ¹⁰⁹ white blood cells from a single leukapheresis to produce CART-19 T cells. Baseline blood leukocytes were also obtained for FDA review needs and studies and cryopreserved. The cellular product is expected to be ready for release after about 2-4 weeks. Flow cytometry quantifies lymphocyte subsets, including CD19 and CD20 B cell assays. Baseline assessment of human anti-VSV-G and anti-mouse antibodies (HAMA). Such cells can be used as a source of cells for the production of CART-19 if the individual has previously accumulated sufficient clearance collections at the Clinical Cell and Vaccine Production Facility in accordance with current good manufacturing practices. The use of accumulated decontamination products will avoid the expense, time and risk of additional scavenging collection by the individual.

Cytoreductive chemotherapy

Lymphocyte depletion chemotherapy will be a high dose of melphalan as described herein.

CART-19 infusion

The infusion will begin on days +12-14 after the stem cells are reinfused.

On days +12-14 prior to the first infusion, the patient will have a differential CBC and be assessed for CD3, CD4, and CD8 numbers, as chemotherapy is partially administered to induce lymphopenia.

The first dose will be administered using a single dose. The cells are thawed at the patient's bedside. Thawed cells will be administered at a rate that is allowed to be fast, such that the infusion duration will be about 10-15 minutes. To facilitate mixing, cells are administered simultaneously using a Y-type adapter. The individual will be infused and pre-medicated as described herein. The vital signs of the individual will be assessed and the pulsating oxygen method will be performed prior to dosing, at the end of the infusion, and every 15 minutes thereafter for 1 hour, and until such stability and satisfaction. Blood samples for determining baseline CART-19 levels were obtained at any time prior to the first infusion and 20 minutes to 4 hours after each infusion (and delivery to TCSL).

result

Three consecutive patients with refractory, advanced multiple myeloma were treated with CTL019 in this ongoing trial. The results of two of these patients were demonstrated based on a primary efficacy assessment at the three-month time point, both of which had substantial anti-tumor effects from CTL019 therapy. The third patient has not reached the three-month time point. The results of the two patients are described in more detail below.

The first myeloma patient has completed its +100 day response assessment and has an excellent response to CART19 therapy. Perform the following tests and the results are as follows:

-SPEP/immunization fixation: negative

- Urine-immune fixation: an immeasurable κ light chain strip that is immuno-fixed (also present on day 38, so non-new)

In other respects, patients meet the criteria for strict complete remission, including the following:

- serum free light chain ratio: normal

- Bone marrow biopsy: negative

-IgA immunophenotype: IgA is below the detection limit

Except for the meager unmeasurable κ light chain results from urinary immunofixation, patients met all criteria for "strict complete remission." A summary of the plasma cell immunophenotype at 3 time points (Day-2, Day +38, Day +103) is shown in Figure 39 and confirms that the patient's IgA is below the detection limit. The summary shows that the heavy chain myeloma load on day -2 was undetectable on days +38 and +103, and the patient was classified as "MRD negative" by flow analysis. On day +103, an overview of normal, multi-strain, CD19+ plasma cells and B cell recovery is shown. The patient has no symptoms of disease or therapy or is generally active as a normal person.

The second patient has not reached the +100 day time point. However, at this time it is proceeding well, but it is too early to determine the effect of CTL019 infusion.

Equivalent

The disclosures of each of the patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety. While the invention has been described with respect to the specific embodiments of the present invention, it will be understood that The scope of the accompanying claims is intended to be understood to include all such aspects and equivalents.

<110> Swiss Business Novartis Corporation University of Pennsylvania Board of Directors

<120> Treatment of cancer with a humanized anti-BCMA chimeric antigen receptor

<130> N2067-7045WO3

<140>

<141>

<150> PCT/CN2014/082586

<151> 2014-07-21

<150> PCT/CN2014/090501

<151> 2014-11-06

<160> 1103

<170> PatentIn version 3.5

<210> 1

<211> 21

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1

<210> 2

<211> 45

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 2

<210> 3

<211> 230

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 3

<210> 4

<211> 282

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 4

<210> 5

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 5

<210> 6

<211> 24

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 6

<210> 7

<211> 42

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 7

<210> 8

<211> 48

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 8

<210> 9

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 9

<210> 10

<211> 112

<212> PRT

<213> Homo sapiens

<400> 10

<210> 11

<211> 1184

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 11

<210> 12

<211> 63

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 12

<210> 13

<211> 135

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 13

<210> 14

<211> 690

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 14

<210> 15

<211> 847

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 15

<210> 16

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 16

<210> 17

<211> 72

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 17

<210> 18

<211> 126

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 18

<210> 19

<211> 123

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 19

<210> 20

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 20

<210> 21

<211> 336

<212> DNA

<213> Homo sapiens

<400> 21

<210> 22

<211> 373

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 22

<210> 23

<211> 1182

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 23

<210> 24

<211> 394

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 24

<210> 25

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 25

<210> 26

<211> 30

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<220>

<221> misc_feature

<222> (1)..(30)

<223> /Remarks="This sequence can cover 1-6 'Gly Gly Gly Gly Ser' repeats Units, some of which may not exist"

<220>

<221> source

<223> /Remarks = "For a detailed description of the substitution and a preferred embodiment, please refer to the submitted manual"

<400> 26

<210> 27

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 27

<210> 28

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 28

<210> 29

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 29

<210> 30

<211> 5000

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<220>

<221> misc_feature

<222> (1)..(5000)

<223> /Remarks="This sequence can cover 50-5,000 nucleotides, some of which may not exist"

<220>

<221> source

<223> /Remarks = "For a detailed description of the substitution and a preferred embodiment, please refer to the submitted manual"

<400> 30

<210> 31

<211> 100

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 31

<210> 32

<211> 5000

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<220>

<221> misc_feature

<222> (1)..(5000)

<223> /Remarks="This sequence can cover 50-5,000 nucleotides, some of which may not exist"

<400> 32

<210> 33

<211> 5000

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<220>

<221> misc_feature

<222> (1)..(5000)

<223> /Remarks="This sequence can cover 100-5,000 nucleotides, some of which may not exist"

<400> 33

<210> 34

<211> 400

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<220>

<221> misc_feature

<222> (1)..(400)

<223> /Remarks="This sequence can cover 100-400 nucleotides, some of which may not exist"

<220>

<221> source

<223> /Remarks = "For a detailed description of the substitution and a preferred embodiment, please refer to the submitted manual"

<400> 34

<210> 35

<211> 2000

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<220>

<221> misc_feature

<222> (1)..(2000)

<223> /Remarks="This sequence can cover 50-2,000 nucleotides, some of which may not exist"

<400> 35

<210> 36

<211> 230

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 36

<210> 37

<211> 690

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 37

<210> 38

<211> 40

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<220>

<221> misc_feature

<222> (1)..(40)

<223> /Remarks="This sequence can cover 1-10 'Gly Gly Gly Ser' repeat units, some of which may not exist"

<220>

<221> source

<223> /Remarks = "For a detailed description of the substitution and a preferred embodiment, please refer to the submitted manual"

<400> 38

<210> 39

<211> 246

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 39

<210> 40

<211> 244

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 40

<210> 41

<211> 243

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 41

<210> 42

<211> 249

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 42

<210> 43

<211> 246

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 43

<210> 44

<211> 247

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 44

<210> 45

<211> 238

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 45

<210> 46

<211> 239

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 46

<210> 47

<211> 241

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 47

<210> 48

<211> 239

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 48

<210> 49

<211> 239

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 49

<210> 50

<211> 246

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 50

<210> 51

<211> 239

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 51

<210> 52

<211> 240

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 52

<210> 53

<211> 241

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 53

<210> 54

<211> 738

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 54

<210> 55

<211> 732

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 55

<210> 56

<211> 729

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 56

<210> 57

<211> 747

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 57

<210> 58

<211> 738

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 58

<210> 59

<211> 741

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 59

<210> 60

<211> 714

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 60

<210> 61

<211> 717

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 61

<210> 62

<211> 723

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 62

<210> 63

<211> 717

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 63

<210> 64

<211> 717

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 64

<210> 65

<211> 738

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 65

<210> 66

<211> 717

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 66

<210> 67

<211> 720

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 67

<210> 68

<211> 723

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 68

<210> 69

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 69

<210> 70

<211> 115

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 70

<210> 71

<211> 115

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 71

<210> 72

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 72

<210> 73

<211> 123

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 73

<210> 74

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 74

<210> 75

<211> 115

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 75

<210> 76

<211> 115

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 76

<210> 77

<211> 115

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 77

<210> 78

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 78

<210> 79

<211> 115

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 79

<210> 80

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 80

<210> 81

<211> 115

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 81

<210> 82

<211> 115

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 82

<210> 83

<211> 115

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 83

<210> 84

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 84

<210> 85

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 85

<210> 86

<211> 111

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 86

<210> 87

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 87

<210> 88

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 88

<210> 89

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 89

<210> 90

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 90

<210> 91

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 91

<210> 92

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 92

<210> 93

<211> 105

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 93

<210> 94

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 94

<210> 95

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 95

<210> 96

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 96

<210> 97

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 97

<210> 98

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 98

<210> 99

<211> 490

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 99

<210> 100

<211> 488

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 100

<210> 101

<211> 487

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 101

<210> 102

<211> 493

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 102

<210> 103

<211> 490

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 103

<210> 104

<211> 491

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 104

<210> 105

<211> 482

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 105

<210> 106

<211> 483

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 106

<210> 107

<211> 485

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 107

<210> 108

<211> 483

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 108

<210> 109

<211> 483

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 109

<210> 110

<211> 490

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 110

<210> 111

<211> 483

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 111

<210> 112

<211> 484

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 112

<210> 113

<211> 485

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 113

<210> 114

<211> 1470

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 114

<210> 115

<211> 1464

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 115

<210> 116

<211> 1461

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 116

<210> 117

<211> 1479

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 117

<210> 118

<211> 1470

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 118

<210> 119

<211> 1473

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 119

<210> 120

<211> 1446

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 120

<210> 121

<211> 1449

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 121

<210> 122

<211> 1455

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 122

<210> 123

<211> 1449

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 123

<210> 124

<211> 1449

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 124

<210> 125

<211> 1470

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 125

<210> 126

<211> 1449

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 126

<210> 127

<211> 1452

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 127

<210> 128

<211> 1455

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 128

<210> 129

<211> 243

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 129

<210> 130

<211> 244

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 130

<210> 131

<211> 246

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 131

<210> 132

<211> 240

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 132

<210> 133

<211> 241

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 133

<210> 134

<211> 245

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 134

<210> 135

<211> 253

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 135

<210> 136

<211> 248

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 136

<210> 137

<211> 246

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 137

<210> 138

<211> 241

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 138

<210> 139

<211> 248

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 139

<210> 140

<211> 248

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 140

<210> 141

<211> 239

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 141

<210> 142

<211> 246

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 142

<210> 143

<211> 241

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 143

<210> 144

<211> 242

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 144

<210> 145

<211> 248

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 145

<210> 146

<211> 244

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 146

<210> 147

<211> 244

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 147

<210> 148

<211> 251

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 148

<210> 149

<211> 246

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 149

<210> 150

<211> 729

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 150

<210> 151

<211> 735

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 151

<210> 152

<211> 738

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 152

<210> 153

<211> 720

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 153

<210> 154

<211> 723

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 154

<210> 155

<211> 735

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 155

<210> 156

<211> 759

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 156

<210> 157

<211> 744

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 157

<210> 158

<211> 738

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 158

<210> 159

<211> 723

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 159

<210> 160

<211> 744

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 160

<210> 161

<211> 744

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 161

<210> 162

<211> 717

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 162

<210> 163

<211> 738

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 163

<210> 164

<211> 723

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 164

<210> 165

<211> 726

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 165

<210> 166

<211> 744

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 166

<210> 167

<211> 732

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 167

<210> 168

<211> 733

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 168

<210> 169

<211> 753

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 169

<210> 170

<211> 739

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 170

<210> 171

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 171

<210> 172

<211> 123

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 172

<210> 173

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 173

<210> 174

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 174

<210> 175

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 175

<210> 176

<211> 123

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 176

<210> 177

<211> 129

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 177

<210> 178

<211> 125

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 178

<210> 179

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 179

<210> 180

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 180

<210> 181

<211> 124

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 181

<210> 182

<211> 124

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 182

<210> 183

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 183

<210> 184

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 184

<210> 185

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 185

<210> 186

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 186

<210> 187

<211> 124

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 187

<210> 188

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 188

<210> 189

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 189

<210> 190

<211> 126

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 190

<210> 191

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 191

<210> 192

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 192

<210> 193

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 193

<210> 194

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 194

<210> 195

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 195

<210> 196

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 196

<210> 197

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 197

<210> 198

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 198

<210> 199

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 199

<210> 200

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 200

<210> 201

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 201

<210> 202

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 202

<210> 203

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 203

<210> 204

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 204

<210> 205

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 205

<210> 206

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 206

<210> 207

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 207

<210> 208

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 208

<210> 209

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 209

<210> 210

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 210

<210> 211

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 211

<210> 212

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 212

<210> 213

<211> 487

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 213

<210> 214

<211> 489

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 214

<210> 215

<211> 490

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 215

<210> 216

<211> 484

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 216

<210> 217

<211> 485

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 217

<210> 218

<211> 489

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 218

<210> 219

<211> 497

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 219

<210> 220

<211> 492

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 220

<210> 221

<211> 490

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 221

<210> 222

<211> 485

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 222

<210> 223

<211> 492

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 223

<210> 224

<211> 492

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 224

<210> 225

<211> 483

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 225

<210> 226

<211> 490

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 226

<210> 227

<211> 485

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 227

<210> 228

<211> 486

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 228

<210> 229

<211> 492

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 229

<210> 230

<211> 488

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 230

<210> 231

<211> 488

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 231

<210> 232

<211> 495

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 232

<210> 233

<211> 490

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 233

<210> 234

<211> 1461

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 234

<210> 235

<211> 1467

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 235

<210> 236

<211> 1470

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 236

<210> 237

<211> 1452

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 237

<210> 238

<211> 1455

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 238

<210> 239

<211> 1467

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 239

<210> 240

<211> 1491

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 240

<210> 241

<211> 1476

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 241

<210> 242

<211> 1470

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 242

<210> 243

<211> 1455

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 243

<210> 244

<211> 1476

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 244

<210> 245

<211> 1476

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 245

<210> 246

<211> 1449

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 246

<210> 247

<211> 1470

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 247

<210> 248

<211> 1455

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 248

<210> 249

<211> 1458

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 249

<210> 250

<211> 1476

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 250

<210> 251

<211> 1464

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 251

<210> 252

<211> 1464

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 252

<210> 253

<211> 1485

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 253

<210> 254

<211> 1470

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 254

<210> 255

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 255

<210> 256

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 256

<210> 257

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 257

<210> 258

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 258

<210> 259

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 259

<210> 260

<211> 111

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 260

<210> 261

<211> 111

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 261

<210> 262

<211> 111

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 262

<210> 263

<211> 244

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 263

<210> 264

<211> 249

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 264

<210> 265

<211> 243

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 265

<210> 266

<211> 243

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 266

<210> 267

<211> 467

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 267

<210> 268

<211> 472

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 268

<210> 269

<211> 466

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 269

<210> 270

<211> 466

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 270

<210> 271

<211> 249

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 271

<210> 272

<211> 747

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 272

<210> 273

<211> 249

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 273

<210> 274

<211> 747

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 274

<210> 275

<211> 132

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 275

<210> 276

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 276

<210> 277

<211> 93

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 277

<210> 278

<211> 95

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 278

<210> 279

<211> 95

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 279

<210> 280

<211> 95

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 280

<210> 281

<211> 95

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Any amino acid

<220>

<221> MOD_RES

<222> (78)..(78)

<223> Any amino acid

<400> 281

<210> 282

<211> 95

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 282

<210> 283

<211> 95

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 283

<210> 284

<211> 1132

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of artificial sequence: synthetic peptide"

<400> 284

<210> 285

<211> 4027

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 285

<210> 286

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 286

<210> 287

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 287

<210> 288

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 288

<210> 289

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 289

<210> 290

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 290

<210> 291

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 291

<210> 292

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 292

<210> 293

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 293

<210> 294

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 294

<210> 295

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 295

<210> 296

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 296

<210> 297

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 297

<210> 298

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 298

<210> 299

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 299

<210> 300

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 300

<210> 301

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 301

<210> 302

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 302

<210> 303

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 303

<210> 304

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 304

<210> 305

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 305

<210> 306

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 306

<210> 307

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 307

<210> 308

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 308

<210> 309

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 309

<210> 310

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 310

<210> 311

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 311

<210> 312

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 312

<210> 313

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 313

<210> 314

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 314

<210> 315

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 315

<210> 316

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 316

<210> 317

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 317

<210> 318

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 318

<210> 319

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 319

<210> 320

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 320

<210> 321

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 321

<210> 322

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 322

<210> 323

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 323

<210> 324

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 324

<210> 325

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 325

<210> 326

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 326

<210> 327

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 327

<210> 328

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 328

<210> 329

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 329

<210> 330

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 330

<210> 331

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 331

<210> 332

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 332

<210> 333

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 333

<210> 334

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 334

<210> 335

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 335

<210> 336

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 336

<210> 337

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 337

<210> 338

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 338

<210> 339

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 339

<210> 340

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 340

<210> 341

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 341

<210> 342

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 342

<210> 343

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 343

<210> 344

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 344

<210> 345

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 345

<210> 346

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 346

<210> 347

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 347

<210> 348

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 348

<210> 349

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 349

<210> 350

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 350

<210> 351

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 351

<210> 352

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 352

<210> 353

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 353

<210> 354

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 354

<210> 355

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 355

<210> 356

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 356

<210> 357

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 357

<210> 358

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 358

<210> 359

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 359

<210> 360

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 360

<210> 361

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 361

<210> 362

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 362

<210> 363

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 363

<210> 364

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 364

<210> 365

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 365

<210> 366

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 366

<210> 367

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 367

<210> 368

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 368

<210> 369

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 369

<210> 370

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 370

<210> 371

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 371

<210> 372

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 372

<210> 373

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 373

<210> 374

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 374

<210> 375

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 375

<210> 376

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 376

<210> 377

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 377

<210> 378

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 378

<210> 379

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 379

<210> 380

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 380

<210> 381

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Oligonucleotide"

<400> 381

<210> 382

<211> 150

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequence: Synthetic Polynucleotide"

<400> 382

<210> 383

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 383

<210> 384

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 384

<210> 385

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 385

<210> 386

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 386

<210> 387

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 387

<210> 388

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 388

<210> 389

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 389

<210> 390

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 390

<210> 391

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 391

<210> 392

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 392

<210> 393

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 393

<210> 394

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 394

<210> 395

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 395

<210> 396

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 396

<210> 397

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 397

<210> 398

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 398

<210> 399

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 399

<210> 400

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 400

<210> 401

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 401

<210> 402

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 402

<210> 403

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 403

<210> 404

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 404

<210> 405

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 405

<210> 406

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 406

<210> 407

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 407

<210> 408

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 408

<210> 409

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 409

<210> 410

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 410

<210> 411

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 411

<210> 412

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 412

<210> 413

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 413

<210> 414

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 414

<210> 415

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 415

<210> 416

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 416

<210> 417

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 417

<210> 418

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 418

<210> 419

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 419

<210> 420

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 420

<210> 421

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 421

<210> 422

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 422

<210> 423

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 423

<210> 424

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 424

<210> 425

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 425

<210> 426

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 426

<210> 427

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 427

<210> 428

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 428

<210> 429

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 429

<210> 430

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 430

<210> 431

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 431

<210> 432

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 432

<210> 433

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 433

<210> 434

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 434

<210> 435

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 435

<210> 436

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 436

<210> 437

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 437

<210> 438

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 438

<210> 439

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 439

<210> 440

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 440

<210> 441

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 441

<210> 442

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 442

<210> 443

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 443

<210> 444

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 444

<210> 445

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 445

<210> 446

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 446

<210> 447

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 447

<210> 448

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 448

<210> 449

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 449

<210> 450

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 450

<210> 451

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 451

<210> 452

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 452

<210> 453

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 453

<210> 454

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 454

<210> 455

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 455

<210> 456

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 456

<210> 457

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 457

<210> 458

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 458

<210> 459

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 459

<210> 460

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 460

<210> 461

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 461

<210> 462

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 462

<210> 463

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 463

<210> 464

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 464

<210> 465

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 465

<210> 466

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 466

<210> 467

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 467

<210> 468

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 468

<210> 469

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 469

<210> 470

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 470

<210> 471

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 471

<210> 472

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 472

<210> 473

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 473

<210> 474

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 474

<210> 475

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 475

<210> 476

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 476

<210> 477

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 477

<210> 478

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> //Remarks = "Description of artificial sequence: synthetic peptide"

<400> 478

<210> 479

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 479

<210> 480

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 480

<210> 481

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 481

<210> 482

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 482

<210> 483

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 483

<210> 484

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 484

<210> 485

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 485

<210> 486

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 486

<210> 487

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 487

<210> 488

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 488

<210> 489

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 489

<210> 490

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 490

<210> 491

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 491

<210> 492

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 492

<210> 493

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 493

<210> 494

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 494

<210> 495

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 495

<210> 496

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 496

<210> 497

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 497

<210> 498

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 498

<210> 499

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 499

<210> 500

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 500

<210> 501

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 501

<210> 502

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 502

<210> 503

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 503

<210> 504

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 504

<210> 505

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 505

<210> 506

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 506

<210> 507

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 507

<210> 508

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 508

<210> 509

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 509

<210> 510

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 510

<210> 511

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 511

<210> 512

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 512

<210> 513

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 513

<210> 514

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 514

<210> 515

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 515

<210> 516

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 516

<210> 517

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 517

<210> 518

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 518

<210> 519

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 519

<210> 520

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 520

<210> 521

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 521

<210> 522

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 522

<210> 523

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 523

<210> 524

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 524

<210> 525

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 525

<210> 526

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 526

<210> 527

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 527

<210> 528

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 528

<210> 529

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 529

<210> 530

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 530

<210> 531

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 531

<210> 532

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 532

<210> 533

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 533

<210> 534

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 534

<210> 535

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 535

<210> 536

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 536

<210> 537

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 537

<210> 538

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 538

<210> 539

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 539

<210> 540

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 540

<210> 541

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 541

<210> 542

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 542

<210> 543

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 543

<210> 544

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 544

<210> 545

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 545

<210> 546

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 546

<210> 547

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 547

<210> 548

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 548

<210> 549

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 549

<210> 550

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 550

<210> 551

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 551

<210> 552

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 552

<210> 553

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 553

<210> 554

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 554

<210> 555

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 555

<210> 556

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 556

<210> 557

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 557

<210> 558

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 558

<210> 559

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 559

<210> 560

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 560

<210> 561

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 561

<210> 562

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 562

<210> 563

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 563

<210> 564

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 564

<210> 565

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 565

<210> 566

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 566

<210> 567

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 567

<210> 568

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 568

<210> 569

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 569

<210> 570

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 570

<210> 571

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 571

<210> 572

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 572

<210> 573

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 573

<210> 574

<211> 叮7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 574

<210> 575

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 575

<210> 576

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 576

<210> 577

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 577

<210> 578

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 578

<210> 579

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 579

<210> 580

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 580

<210> 581

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 581

<210> 582

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 582

<210> 583

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 583

<210> 584

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 584

<210> 585

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 585

<210> 586

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 586

<210> 587

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 587

<210> 588

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 588

<210> 589

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 589

<210> 590

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 590

<210> 591

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 591

<210> 592

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 592

<210> 593

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 593

<210> 594

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 594

<210> 595

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 595

<210> 596

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 596

<210> 597

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 597

<210> 598

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 598

<210> 599

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 599

<210> 600

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 600

<210> 601

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 601

<210> 602

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 602

<210> 603

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 603

<210> 604

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 604

<210> 605

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 605

<210> 606

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 606

<210> 607

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 607

<210> 608

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 608

<210> 609

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 609

<210> 610

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 610

<210> 611

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 611

<210> 612

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 612

<210> 613

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 613

<210> 614

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 614

<210> 615

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 615

<210> 616

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 616

<210> 617

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 617

<210> 618

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 618

<210> 619

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 619

<210> 620

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 620

<210> 621

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 621

<210> 622

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 622

<210> 623

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 623

<210> 624

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 624

<210> 625

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 625

<210> 626

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 626

<210> 627

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 627

<210> 628

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 628

<210> 629

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 629

<210> 630

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 630

<210> 631

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 631

<210> 632

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 632

<210> 633

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 633

<210> 634

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 634

<210> 635

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 635

<210> 636

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 636

<210> 637

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 637

<210> 638

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks="" Description of Artificial Sequence: Synthetic Peptides"

<400> 638

<210> 639

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 639

<210> 640

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 640

<210> 641

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 641

<210> 642

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 642

<210> 643

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 643

<210> 644

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 644

<210> 645

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 645

<210> 646

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 646

<210> 647

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 647

<210> 648

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 648

<210> 649

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 649

<210> 650

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 650

<210> 651

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 651

<210> 652

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 652

<210> 653

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 653

<210> 654

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 654

<210> 655

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 655

<210> 656

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 656

<210> 657

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 657

<210> 658

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 658

<210> 659

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 659

<210> 660

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 660

<210> 661

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 661

<210> 662

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 662

<210> 663

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 663

<210> 664

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 664

<210> 665

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 665

<210> 666

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 666

<210> 667

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 667

<210> 668

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 668

<210> 669

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 669

<210> 670

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 670

<210> 671

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 671

<210> 672

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 672

<210> 673

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 673

<210> 674

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 674

<210> 675

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 675

<210> 676

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 676

<210> 677

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 677

<210> 678

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 678

<210> 679

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 679

<210> 680

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 680

<210> 681

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 681

<210> 682

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 682

<210> 683

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 683

<210> 684

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 684

<210> 685

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 685

<210> 686

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 686

<210> 687

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 687

<210> 688

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 688

<210> 689

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 689

<210> 690

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 690

<210> 691

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 691

<210> 692

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 692

<210> 693

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 693

<210> 694

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 694

<210> 695

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 695

<210> 696

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 696

<210> 697

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 697

<210> 698

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 698

<210> 699

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 699

<210> 700

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 700

<210> 701

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 701

<210> 702

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 702

<210> 703

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 703

<210> 704

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 704

<210> 705

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 705

<210> 706

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 706

<210> 707

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 707

<210> 708

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 708

<210> 709

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 709

<210> 710

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 710

<210> 711

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 711

<210> 712

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 712

<210> 713

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 713

<210> 714

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 714

<210> 715

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 715

<210> 716

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 716

<210> 717

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 717

<210> 718

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 718

<210> 719

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 719

<210> 720

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 720

<210> 721

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 721

<210> 722

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 722

<210> 723

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 723

<210> 724

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 724

<210> 725

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 725

<210> 726

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 726

<210> 727

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 727

<210> 728

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 728

<210> 729

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 729

<210> 730

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 730

<210> 731

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 731

<210> 732

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 732

<210> 733

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 733

<210> 734

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 734

<210> 735

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 735

<210> 736

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 736

<210> 737

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 737

<210> 738

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 738

<210> 739

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 739

<210> 740

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 740

<210> 741

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 741

<210> 742

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 742

<210> 743

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 743

<210> 744

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 744

<210> 745

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 745

<210> 746

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 746

<210> 747

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 747

<210> 748

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 748

<210> 749

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 749

<210> 750

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 750

<210> 751

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 751

<210> 752

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 752

<210> 753

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 753

<210> 754

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 754

<210> 755

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 755

<210> 756

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 756

<210> 757

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 757

<210> 758

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 758

<210> 759

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 759

<210> 760

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 760

<210> 761

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 761

<210> 762

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 762

<210> 763

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 763

<210> 764

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 764

<210> 765

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 765

<210> 766

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 766

<210> 767

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 767

<210> 768

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 768

<210> 769

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 769

<210> 770

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 770

<210> 771

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 771

<210> 772

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 772

<210> 773

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 773

<210> 774

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 774

<210> 775

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 775

<210> 776

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 776

<210> 777

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 777

<210> 778

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 778

<210> 779

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 779

<210> 780

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 780

<210> 781

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 781

<210> 782

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 782

<210> 783

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 783

<210> 784

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 784

<210> 785

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 785

<210> 786

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 786

<210> 787

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 787

<210> 788

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 788

<210> 789

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 789

<210> 790

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 790

<210> 791

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 791

<210> 792

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 792

<210> 793

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 793

<210> 794

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 794

<210> 795

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 795

<210> 796

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 796

<210> 797

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 797

<210> 798

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 798

<210> 799

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 799

<210> 800

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 800

<210> 801

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 801

<210> 802

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 802

<210> 803

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 803

<210> 804

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 804

<210> 805

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 805

<210> 806

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 806

<210> 807

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 807

<210> 808

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 808

<210> 809

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 809

<210> 810

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 810

<210> 811

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 811

<210> 812

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 812

<210> 813

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 813

<210> 814

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 814

<210> 815

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 815

<210> 816

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 816

<210> 817

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 817

<210> 818

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 818

<210> 819

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 819

<210> 820

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 820

<210> 821

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 821

<210> 822

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 822

<210> 823

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 823

<210> 824

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 824

<210> 825

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 825

<210> 826

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 826

<210> 827

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 827

<210> 828

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 828

<210> 829

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 829

<210> 830

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 830

<210> 831

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 831

<210> 832

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 832

<210> 833

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 833

<210> 834

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 834

<210> 835

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 835

<210> 836

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 836

<210> 837

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 837

<210> 838

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 838

<210> 839

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 839

<210> 840

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 840

<210> 841

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 841

<210> 842

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 842

<210> 843

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 843

<210> 844

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 844

<210> 845

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 845

<210> 846

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 846

<210> 847

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 847

<210> 848

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 848

<210> 849

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 849

<210> 850

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 850

<210> 851

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 851

<210> 852

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 852

<210> 853

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 853

<210> 854

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 854

<210> 855

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 855

<210> 856

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 856

<210> 857

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 857

<210> 858

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 858

<210> 859

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 859

<210> 860

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 860

<210> 861

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 861

<210> 862

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 862

<210> 863

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 863

<210> 864

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 864

<210> 865

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 865

<210> 866

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 866

<210> 867

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 867

<210> 868

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 868

<210> 869

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 869

<210> 870

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 870

<210> 871

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 871

<210> 872

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 872

<210> 873

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 873

<210> 874

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 874

<210> 875

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 875

<210> 876

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 876

<210> 877

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 877

<210> 878

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 878

<210> 879

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 879

<210> 880

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 880

<210> 881

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 881

<210> 882

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 882

<210> 883

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 883

<210> 884

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 884

<210> 885

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 885

<210> 886

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 886

<210> 887

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 887

<210> 888

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 888

<210> 889

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 889

<210> 890

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 890

<210> 891

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 891

<210> 892

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 892

<210> 893

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 893

<210> 894

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 894

<210> 895

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 895

<210> 896

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 896

<210> 897

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 897

<210> 898

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 898

<210> 899

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 899

<210> 900

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 900

<210> 901

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 901

<210> 902

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 902

<210> 903

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 903

<210> 904

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 904

<210> 905

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 905

<210> 906

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 906

<210> 907

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 907

<210> 908

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 908

<210> 909

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 909

<210> 910

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 910

<210> 911

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 911

<210> 912

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 912

<210> 913

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 913

<210> 914

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 914

<210> 915

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 915

<210> 916

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 916

<210> 917

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 917

<210> 918

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 918

<210> 919

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 919

<210> 920

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 920

<210> 921

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 921

<210> 922

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 922

<210> 923

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 923

<210> 924

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 924

<210> 925

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 925

<210> 926

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 926

<210> 927

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 927

<210> 928

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 928

<210> 929

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 929

<210> 930

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 930

<210> 931

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 931

<210> 932

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 932

<210> 933

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 933

<210> 934

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 934

<210> 935

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 935

<210> 936

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 936

<210> 937

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 937

<210> 938

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 938

<210> 939

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 939

<210> 940

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 940

<210> 941

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 941

<210> 942

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 942

<210> 943

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 943

<210> 944

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 944

<210> 945

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 945

<210> 946

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 946

<210> 947

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 947

<210> 948

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 948

<210> 949

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 949

<210> 950

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 950

<210> 951

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 951

<210> 952

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 952

<210> 953

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 953

<210> 954

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 954

<210> 955

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 955

<210> 956

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 956

<210> 957

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 957

<210> 958

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 958

<210> 959

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 959

<210> 960

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 960

<210> 961

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 961

<210> 962

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 962

<210> 963

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 963

<210> 964

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 964

<210> 965

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 965

<210> 966

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 966

<210> 967

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 967

<210> 968

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 968

<210> 969

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 969

<210> 970

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 970

<210> 971

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 971

<210> 972

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 972

<210> 973

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 973

<210> 974

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 974

<210> 975

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 975

<210> 976

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 976

<210> 977

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 977

<210> 978

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 978

<210> 979

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 979

<210> 980

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 980

<210> 981

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 981

<210> 982

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 982

<210> 983

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 983

<210> 984

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 984

<210> 985

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 985

<210> 986

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 986

<210> 987

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 987

<210> 988

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 988

<210> 989

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 989

<210> 990

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 990

<210> 991

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 991

<210> 992

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 992

<210> 993

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 993

<210> 994

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 994

<210> 995

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 995

<210> 996

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 996

<210> 997

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 997

<210> 998

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 998

<210> 999

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 999

<210> 1000

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1000

<210> 1001

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1001

<210> 1002

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1002

<210> 1003

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1003

<210> 1004

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1004

<210> 1005

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1005

<210> 1006

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1006

<210> 1007

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1007

<210> 1008

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1008

<210> 1009

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1009

<210> 1010

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1010

<210> 1011

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1011

<210> 1012

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1012

<210> 1013

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1013

<210> 1014

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1014

<210> 1015

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1015

<210> 1016

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1016

<210> 1017

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1017

<210> 1018

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1018

<210> 1019

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1019

<210> 1020

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1020

<210> 1021

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1021

<210> 1022

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1022

<210> 1023

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1023

<210> 1024

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1024

<210> 1025

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1025

<210> 1026

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1026

<210> 1027

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1027

<210> 1028

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1028

<210> 1029

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1029

<210> 1030

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1030

<210> 1031

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1031

<210> 1032

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1032

<210> 1033

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1033

<210> 1034

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1034

<210> 1035

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1035

<210> 1036

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1036

<210> 1037

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1037

<210> 1038

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1038

<210> 1039

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1039

<210> 1040

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1040

<210> 1041

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1041

<210> 1042

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1042

<210> 1043

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1043

<210> 1044

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1044

<210> 1045

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1045

<210> 1046

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1046

<210> 1047

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1047

<210> 1048

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1048

<210> 1049

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1049

<210> 1050

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1050

<210> 1051

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1051

<210> 1052

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1052

<210> 1053

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1053

<210> 1054

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1054

<210> 1055

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1055

<210> 1056

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1056

<210> 1057

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1057

<210> 1058

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1058

<210> 1059

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1059

<210> 1060

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1060

<210> 1061

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1061

<210> 1062

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1062

<210> 1063

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1063

<210> 1064

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1064

<210> 1065

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1065

<210> 1066

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1066

<210> 1067

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1067

<210> 1068

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1068

<210> 1069

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1069

<210> 1070

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1070

<210> 1071

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1071

<210> 1072

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1072

<210> 1073

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1073

<210> 1074

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1074

<210> 1075

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1075

<210> 1076

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1076

<210> 1077

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1077

<210> 1078

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1078

<210> 1079

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1079

<210> 1080

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1080

<210> 1081

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1081

<210> 1082

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1082

<210> 1083

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1083

<210> 1084

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1084

<210> 1085

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1085

<210> 1086

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1086

<210> 1087

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1087

<210> 1088

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1088

<210> 1089

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1089

<210> 1090

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1090

<210> 1091

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1091

<210> 1092

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1092

<210> 1093

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1093

<210> 1094

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1094

<210> 1095

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1095

<210> 1096

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1096

<210> 1097

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1097

<210> 1098

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1098

<210> 1099

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1099

<210> 1100

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1100

<210> 1101

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1101

<210> 1102

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1102

<210> 1103

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /Remarks = "Description of Artificial Sequences: Synthetic Peptides"

<400> 1103

Claims (66)

An isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an anti-BCMA binding domain (eg, a human or humanized anti-BCMA binding domain), a transmembrane domain, and intracellular signaling a domain, and wherein the anti-BCMA binding domain comprises the heavy chain complementarity determining region 1 (HC CDR1) of any anti-BMCA heavy chain binding domain amino acid sequence listed in Table 1 or 16, heavy chain complementation determining Region 2 (HC CDR2) and heavy chain complementarity determining region 3 (HC CDR3). The isolated nucleic acid molecule of claim 1, wherein the anti-BCMA binding domain further comprises a light chain complementarity determining region 1 of any anti-BMCA light chain binding domain amino acid sequence set forth in Table 1 or 16 (LC) CDR1), light chain complementarity determining region 2 (LC CDR2) and light chain complementarity determining region 3 (LC CDR3). The isolated nucleic acid molecule of claim 2, wherein the LC CDR1, LC CDR2 and LC CDR3 are the LC CDR sequences set forth in Tables 21, 23 or 25. The isolated nucleic acid molecule of claims 1 to 3, wherein the HC CDR1, HC CDR2 and HC CDR3 are the HC CDR sequences set forth in Tables 20, 22 or 24. The isolated nucleic acid molecule of any one of claims 1 to 4, which encodes an amino acid sequence comprising: CAR: (i) any of the light chain variable regions listed in Table 1; (ii) having a pair of tables At least one, two or three modifications of the amino acid sequence of any of the light chain variable regions provided in 1 but no more than 30, 20 or 10 modified amino acid sequences; or Iii) an amino acid sequence having 95%-99% identity with the amino acid sequence of any of the light chain variable regions provided in Table 1. The isolated nucleic acid molecule of any one of claims 1 to 5, which encodes a CAR comprising: (i) an amino acid sequence of any of the heavy chain variable regions listed in Table 1; (ii) at least one of the amino acid sequences having any of the heavy chain variable regions provided in Table 1 , two or three modified but no more than 30, 20 or 10 modified amino acid sequences; or (iii) amino acids of any of the heavy chain variable regions provided in Table 1 The sequence has an amino acid sequence of 95%-99% identity. The isolated nucleic acid molecule of any one of claims 1 to 6, which encodes an amino acid sequence comprising any of the light chain variable regions listed in Table 1, and any of the heavy chain variable regions listed in Table 1. CAR of the amino acid sequence. The isolated nucleic acid molecule of any of the preceding claims, wherein the encoded anti-BCMA binding domain comprises: (i) selected from the group consisting of SEQ ID NO: 49, 39-53, 69-83, 84-98, Amino acid sequence of the group consisting of 129-149, 171-191, 192-212, 255-258, 259-262, 263-266, 271 or 273; (ii) having pairs 49, 39-53, 69-83 At least one, two or three modifications of any of 84-98, 129-149, 171-191, 192-212, 255-258, 259-262, 263-266, 271 or 273 but no more than 30 , 20 or 10 modified amino acid sequences; or (iii) with 49, 39-53, 69-83, 84-98, 129-149, 171-191, 192-212, 255-258, 259 Any of -262, 263-266, 271 or 273 has a 95%-99% identity amino acid sequence. The isolated nucleic acid molecule of any one of the preceding claims, wherein the anti-BCMA binding domain comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 64, 54-68, 150-170, 272 or 274 Or a sequence with 95%-99% identity. The isolated nucleic acid molecule of any of the preceding claims, wherein: (i) the encoded CAR comprises a transmembrane domain comprising a selected from the group consisting of A transmembrane domain of a protein consisting of a group of alpha, beta or scorpion chains of T cell receptors, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64 CD80, CD86, CD134, CD137 and CD154; (ii) the encoded transmembrane domain comprises the amino acid sequence of SEQ ID NO: 6, comprising at least one of the amino acid sequence of SEQ ID NO: 6, Two or three modified but no more than 20, 10 or 5 modified amino acid sequences, or a sequence having 95%-99% identity to the amino acid sequence of SEQ ID NO: 6; or (ii The nucleic acid sequence encoding the transmembrane domain comprises the sequence of SEQ ID NO: 17, or a sequence with 95%-99% identity thereto. The isolated nucleic acid molecule of any of the preceding claims, wherein the encoded anti-BCMA binding domain comprises: (i) the amino acid sequence of SEQ ID NO: 49; (ii) has the pair SEQ ID NO: At least one, two or three modifications of 49 but no more than 30, 20 or 10 modified amino acid sequences; or (iii) an amine group having 95% to 99% identity to SEQ ID NO: 49 Acid sequence. The isolated nucleic acid molecule of any of the preceding claims, wherein the encoded anti-BCMA binding domain is joined to the transmembrane domain by a hinge region. The isolated nucleic acid molecule of claim 12, wherein (i) the encoded hinge region comprises an amino acid sequence of SEQ ID NO: 2 or a sequence having 95%-99% identity thereto; or (ii) encoding the The nucleic acid sequence of the hinge region comprises the nucleotide sequence of SEQ ID NO: 13 or a sequence with 95% to 99% identity thereto. The isolated nucleic acid molecule of any one of the preceding claims, wherein the encoded costimulatory domain is a functional signaling domain obtained from a protein selected from the group consisting of: MHC class I molecules, TNF receptors Body protein, immunoglobulin White-like protein, cytokine receptor, integrin, signaling lymphocyte activating molecule (SLAM protein), activated NK cell receptor, BTLA, Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD , CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226 ), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM ( SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a and ligands that specifically bind to CD83. The isolated nucleic acid molecule of claim 14, wherein the encoded costimulatory domain comprises the amino acid sequence of SEQ ID NO: 7, having at least one, two or of the amino acid sequence of SEQ ID NO: Three modified but no more than 20, 10 or 5 modified amino acid sequences, or a sequence having 95%-99% identity to the amino acid sequence of SEQ ID NO: 7. The isolated nucleic acid molecule of claim 14, wherein the nucleic acid sequence encoding the costimulatory domain comprises the nucleotide sequence of SEQ ID NO: 18 or a sequence having 95% to 99% identity thereto. The isolated nucleic acid molecule of any of the preceding claims, wherein the encoded intracellular signaling domain comprises a functional signaling domain of 4-1BB and/or a functional signaling domain of CD3ξ. The isolated nucleic acid molecule of any of the preceding claims, wherein the encoded intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 7 and/or SEQ ID NO: 9 or SEQ ID NO: 10. a sequence; or having at least one, two or three modifications to the amino acid sequence of SEQ ID NO: 7 and/or the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10 but no more than 20 , 10 or 5 modified amino acid sequences, or 95%-99% with the amino acid sequence of SEQ ID NO: 7 and/or the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10. The sequence of consistency. The isolated nucleic acid molecule of any of the preceding claims, wherein the encoded intracellular signaling domain comprises the sequence of SEQ ID NO: 7 and the sequence of SEQ ID NO: 9 or SEQ ID NO: 10, wherein The sequences of the intracellular signaling domain are represented in the same framework and are in the form of a single polypeptide chain. The isolated nucleic acid molecule according to any one of the preceding claims, wherein the nucleic acid sequence encoding the intracellular signaling domain comprises the sequence of SEQ ID NO: 18 or a sequence having 95%-99% identity thereto, and/or The sequence of SEQ ID NO: 20 or SEQ ID NO: 21, or a sequence with 95% to 99% identity thereto. The isolated nucleic acid molecule of any of the preceding claims, further comprising a leader sequence encoding the amino acid sequence of SEQ ID NO: 1. An isolated nucleic acid molecule according to any one of the preceding claims, which encodes an amino acid sequence comprising CAR: (i) any one of SEQ ID NO: 109, 99-113, 213-233 or 267-270 (ii) having at least one, two or three modifications to any one of SEQ ID NO: 109, 99-113, 213-233 or 267-270 but no more than 30, 20 or 10 repairs A decorated amino acid sequence; or (iii) an amino acid sequence having 95% to 99% identity to any of SEQ ID NO: 109, 99-113, 213-233, or 267-270. An isolated nucleic acid molecule according to any one of the preceding claims, which comprises the nucleotide sequence of any one of SEQ ID NO: 124, 114-128 or 234-254, or SEQ ID NO: 124, 114-128 Or any of 234-254 has a 95%-99% identity nucleotide sequence. An isolated polypeptide molecule encoded by the nucleic acid molecule of any one of claims 1 to 23. An isolated chimeric antigen receptor (CAR) polypeptide, wherein the CAR comprises a human anti-BCMA binding domain, a transmembrane domain, and an intracellular signaling structure comprising a costimulatory domain and/or a major signaling domain An antibody or antibody fragment of a domain, and wherein the anti-BCMA binding domain comprises the heavy chain complementarity determining region 1 (HC CDR1) of any anti-BCMA heavy chain binding domain amino acid sequence set forth in Table 1 or 16, Heavy chain complementarity determining region 2 (HC CDR2) and heavy chain complementarity determining region 3 (HC CDR3). The isolated CAR polypeptide of claim 25, wherein the human anti-BCMA binding domain further comprises a light chain complementarity determining region 1 of any anti-BCMA light chain binding domain amino acid sequence set forth in Table 1 or 16 ( LC CDR1), light chain complementarity determining region 2 (LC CDR2) and light chain complementarity determining region 3 (LC CDR3). The isolated CAR polypeptide of claim 24, wherein the LC CDR1, LC CDR2 and LC CDR3 are the LC CDR sequences set forth in Tables 21, 23 or 25. The isolated CAR polypeptide of any one of clauses 25 to 27, wherein the HC CDR1, HC CDR2 and HC CDR3 are the HC CDR sequences set forth in Tables 21, 23 or 25. The isolated CAR polypeptide of any one of claims 25 to 28, comprising: (i) an amino acid sequence of any of the light chain variable regions listed in Table 1; (ii) having at least one of the amino acid sequences of any of the light chain variable regions provided in Table 1, Two or three modified but no more than 30, 20 or 10 modified amino acid sequences; or (iii) having the amino acid sequence of any of the light chain variable regions provided in Table 1 95%-99% identical amino acid sequence. The isolated CAR polypeptide of any one of claims 25 to 29, comprising: (i) an amino acid sequence of any of the heavy chain variable regions listed in Table 1; (ii) having the properties provided in Table 1 At least one, two or three modifications of the amino acid sequence of any of the heavy chain variable regions but no more than 30, 20 or 10 modified amino acid sequences; or (iii) The amino acid sequence of any of the heavy chain variable regions provided in 1 has a 95%-99% identity amino acid sequence. The isolated CAR polypeptide of any one of claims 25 to 30, which comprises an amino acid sequence of any of the light chain variable regions listed in Table 1 and an amine of any of the heavy chain variable regions provided in Table 1. Base acid sequence. The isolated CAR polypeptide of any one of claims 25 to 31, comprising: (i) selected from the group consisting of 49, 39-53, 69-83, 84-98, 129-149, 171-191, 192-212, Any amino acid sequence of the group consisting of 255-258, 259-262, 263-266, 271, and 273; (ii) having pairs of SEQ ID NOS: 49, 39-53, 69-83, 84-98, 129- At least one, two or three modifications of 149, 171-191, 192-212, 255-258, 259-262, 263-266, 271, and 273 but no more than 30, 20, or 10 a modified amino acid sequence; or (iii) and SEQ ID NO: 49, 39-53, 69-83, 84-98, 129-149, 171-191, 192-212, 255-258, 259-262, Any of 263-266, 271, and 273 Amino acid sequence with 95%-99% identity. The isolated CAR polypeptide of any one of clauses 25 to 32, wherein the transmembrane domain comprises a transmembrane domain from a protein selected from the group consisting of: alpha, beta or purine of a T cell receptor Chain, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154. The isolated CAR polypeptide of any one of clauses 25 to 33, wherein (i) the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 6; (ii) the amino acid sequence comprises SEQ ID NO: At least one, two or three modifications of the amino acid sequence of 6 but no more than 20, 10 or 5 modifications; or (iii) 95%-99% with the amino acid sequence of SEQ ID NO: 6. The sequence of consistency. The isolated CAR polypeptide of any one of clauses 25 to 34, wherein the anti-BCMA binding domain is joined to the transmembrane domain by a hinge region. The isolated CAR polypeptide of claim 35, wherein the hinge region comprises SEQ ID NO: 2 or SEQ ID NO: 36 or a sequence having 95% to 99% identity thereto. The isolated CAR polypeptide of any one of clauses 25 to 36, wherein the costimulatory domain is a functional signaling domain obtained from a protein selected from the group consisting of: MHC class I molecules, TNF receptors Protein, immunoglobulin-like protein, cytokine receptor, integrin, signaling lymphocyte activating molecule (SLAM protein), activated NK cell receptor, BTLA, Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229 ), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a and ligands that specifically bind to CD83. The isolated CAR polypeptide of any one of clauses 25 to 37, wherein the costimulatory domain comprises the amino acid sequence of SEQ ID NO: 7, or has at least one of the amino acid sequence of SEQ ID NO: , two or three modified but no more than 20, 10 or 5 modified amino acid sequences, or a sequence having 95%-99% identity to the amino acid sequence of SEQ ID NO: 7. The isolated CAR polypeptide of any one of clauses 25 to 37, wherein the intracellular signaling domain comprises a functional signaling domain of 4-1BB and/or a functional signaling domain of CD3ξ. The isolated CAR polypeptide of any one of clauses 25 to 39, wherein the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 7 and/or SEQ ID NO: 9 or SEQ ID NO: a sequence, or having at least one, two or three modifications to the amino acid sequence of SEQ ID NO: 7 and/or the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10, but no more than 20, 10 or 5 modified amino acid sequences, or 95%-99% identical to the amino acid sequence of SEQ ID NO: 7 and/or the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10. Sequence of sex. The isolated CAR polypeptide of any one of clauses 25 to 40, wherein the intracellular signaling domain comprises the sequence of SEQ ID NO: 7 and the sequence of SEQ ID NO: 9 or SEQ ID NO: 10, wherein These sequences of the intracellular signaling domain are represented in the same framework and are in the form of a single polypeptide chain. The isolated CAR polypeptide of any one of claims 25 to 41, which further comprises a leader sequence comprising the amino acid sequence of SEQ ID NO: 1. The isolated CAR polypeptide of any one of claims 25 to 42, which comprises: (i) an amino acid sequence of any one of SEQ ID NO: 109, 99-113, 213-233 or 267-270; Ii) an amine group having at least one, two or three modifications but no more than 30, 20 or 10 modifications to any one of SEQ ID NO: 109, 99-113, 213-233 or 267-270 An acid sequence; or (iii) an amino acid sequence having 95% to 99% identity to any of SEQ ID NO: 109, 99-113, 213-233, or 267-270. A vector comprising a nucleic acid molecule encoding a CAR polypeptide according to any of the preceding claims, wherein the vector is selected from the group consisting of a DNA vector, an RNA vector, a plastid, a lentiviral vector, an adenoviral vector or a retroviral vector. group. The vector of claim 44, which further comprises an EF-1 promoter comprising the sequence of SEQ ID NO: 11. A cell, such as an immune effector cell, comprising a nucleic acid according to any one of claims 1 to 23, a CAR polypeptide according to any one of claims 24 to 43 or a vector according to any one of claims 44 or 45. A method of producing a cell, such as an immune effector cell, comprising transducing an immune effector cell with a vector as claimed in claim 44 or 45. A method of producing an RNA engineered cell population comprising introducing an in vitro transcribed RNA or synthetic RNA into a cell, wherein the RNA comprises a coding as in the former A nucleic acid of a CAR polypeptide according to any one of the claims. A method of providing anti-tumor immunity in a mammal, comprising administering to the mammal an effective amount of a cell, such as a population of immune effector cells, comprising the CAR nucleic acid of any one of claims 1 to 23 or as requested The CAR polypeptide of any one of items 24 to 43. The method of claim 49, wherein the cell is an autologous T cell or an allogeneic T cell. A method of treating a mammal having a disease associated with BCMA manifestation comprising administering to the mammal an effective amount of a cell, such as a population of immune effector cells, the cell comprising the CAR of any one of claims 1 to 23 A nucleic acid or a CAR polypeptide according to any one of claims 24 to 43. The method of claim 51, wherein the disease associated with BCMA performance is: (i) cancer or a malignant disease, or a precancerous disease selected from one or more of myelodysplasia, myelodysplastic syndrome, or leukemia prodrosis Or (ii) non-cancer-related indications associated with BCMA performance. The method of claim 51 or 52, wherein the disease is blood cancer. The method of any one of claims 51 to 53, wherein the disease is acute leukemia, which is selected from the group consisting of B cell acute lymphoblastic leukemia ("BALL"), T cell acute lymphocytic leukemia ("TALL"), acute lymphoid Leukemia (ALL); chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL); B-cell pro-lymphocytic leukemia, parental plasmacytoid dendritic cell tumor, Burkitt's lymphoma ( Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative condition, MALT lymphoma, mantle cell lymphoma, margin Lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma, plasmablast lymphoma, plasmacytoid Dendritic cell tumor, Waldenstrom macroglobulinemia; prostate cancer (eg, castration resistant or therapeutic resistant prostate cancer, or metastatic prostate cancer), pancreatic cancer, lung cancer; Plasma cell proliferative disorders (such as asymptomatic myeloma (and suspicion of multiple myeloma or inert myeloma), undefined gamma globulinemia (MGUS), Waldenstrom macroglobulinemia, plasma cells Tumors (eg, plasma cell cachexia, solitary myeloma, solitary plasmacytoma, extramedullary plasmacytoma, and multiple plasmacytoma), systemic amyloid light chain amyloidosis, and POEMS syndrome (also known as Croat) - Crow-Fukase syndrome, Takatsuki disease and PEP syndrome) or a combination thereof. The method of any one of claims 51 to 54, wherein the cell, such as the population of immune effector cells, is administered in combination with one or more of: (i) increasing the cell comprising the CAR nucleic acid or CAR polypeptide An agent that is effective; (ii) an agent that improves one or more of the side effects associated with administering the cell comprising the CAR nucleic acid or the CAR polypeptide; (iii) an agent that treats the BCMA-related disease. The method of any one of claims 51 to 55, wherein the cell, such as a population of immune effector cells, is administered in combination with a PD-L1 inhibitor, such as an anti-PD-L1 antibody. The isolated nucleic acid molecule of any one of claims 1 to 23, the isolated CAR polypeptide molecule of any one of claims 24 to 43, the vector of claim 44 or 45, or the cell of claim 46, It is used as a medicament. The isolated nucleic acid molecule of any one of claims 1 to 23, the isolated CAR polypeptide molecule of any one of claims 24 to 43, the vector of claim 44 or 45, or the cell of claim 46, It is used to treat diseases associated with BCMA performance. The cell of claim 46, such as a population of immune effector cells, further comprising an inhibitory molecule comprising a first plurality comprising at least a portion of the inhibitory molecule A peptide that associates with a second polypeptide comprising a positive signal from an intracellular signaling domain. The cell of claim 59, wherein the inhibitory molecule comprises a first polypeptide comprising at least a portion of PD1 and a second polypeptide comprising a costimulatory domain and a major signaling domain. The method of claim 55, wherein the agent is an mTOR inhibitor and the subject is administered a low immunopotentiating dose of an mTOR inhibitor, such as RAD001 or rapamycin. The method of claim 61, wherein the mTOR inhibitor is administered in an amount sufficient to reduce the proportion of PD-1 positive T cells and increase PD-1 negative in the peripheral blood of the individual or from the T cell preparation isolated from the individual. The ratio of T cells or the ratio of PD-1 negative T cells/PD-1 positive T cells. The method of any one of claims 51 to 56, wherein the cell expressing the CAR molecule is administered in combination with a cell comprising a CD19 CAR molecule. The method of claim 63, wherein the BCMA-related disease is multiple myeloma. The method of claim 64, wherein the multiple myeloma is CD19-negative multiple myeloma. The method of any one of claims 51 to 56 and 63 to 65, wherein the cell line expressing the CAR molecule is administered to the individual in separate doses, for example, once, twice, three times or more in separate doses, Depending on the situation, the first percentage of the total dose is administered on the first day of treatment, on the subsequent treatment day (eg, the second day, the third day, the fourth day, the fifth day, the sixth day, or the seventh day or a later percentage of the total dose is administered, and on a subsequent treatment day (eg, third day, fourth day, fifth day, sixth day, seventh day, eighth day, first Nine days, tenth day or later days, depending on the situation, the third percentage (eg, the remaining percentage) of the total dose is administered, as the case may be, 10% of the total dose of the cells is administered on the first day, in the first two days and 30% of the total administered dose of the cells, and the remaining 60% in the third day of treatment with the total dose of administered cells, and optionally wherein the total cell dose comprises 1 to 5 × 10 ^7, or 1 to 5 × 10 ⁸ cells.